Methods of detecting and isolating a ripening form of a polypeptide having rhamnogalacturonase activity

ABSTRACT

The invention relates to isolation of an Aspergillus gene encoding rhamnogalacturonase (RG-ase) and the construction of recombinant Aspergillus strains with overexpression of RG-ase. These strains can be used for the commercial production of RG-ase. RG-ase is an important enzyme in processes requiring the degradation and/or modification of pectin or modification of pectin-containing vegetable or plant cell wall material. RG-ase may be used in various applications, including the processing of fruits and vegetables, in the extraction of components from vegetable material or for improving the functionality of pectin or pectin-containing vegetable material, food material or plant cell wall material.

This is a division of application Ser. No. 08/061,062, filed May 14,1993.

The present invention relates to the field of recombinant DNA technologyand more in particular relates to its use in view of thebiotechnological production of a polypeptide having rhamnogalacturonaseactivity. A polypeptide having rhamnogalacturonase activity is apolypeptide that can partly degrade pectin molecules and can be used inany context where degradation of pectin molecules is desirable, such asin the processing of fruit and vegetables, in the extraction of foodingredients by degradation of plant cell walls or in brewing processes.

BACKGROUND OF THE INVENTION

During fruit juice manufacture enzyme preparations are often used in thesteps of extraction and liquefaction of fruit and fruit juiceclarification (Voragen 1989). The commercial enzyme preparations containa mixture of mainly pectinases (e.g. polygalacturonases, pectinesterases, pectin transeliminases) together with minor quantities ofother hydrolytic enzymes such as arabinases, galactanases and xylanases.The substrates for the various pectinases are pectins, which arepolygalacturonides of high molecular weight (20000-40000 D) consistingof α-1,4-glycosidic bound D-galacturonic acid polymers. Some of theuronic acid groups are esterified with methanol. The polygalacturonicbackbone is interrupted by so-called hairy regions, consisting of arhamnose-galacturonic acid backbone with arabinose-rich side chains(Voragen and Beldman 1990).

Pectins occur in nature as constituents of higher plant cell walls. Theyare found in the primary cell wall and middle lamella where they areembedded in cellulose fibrils (Mc Neil et al. 1984). The composition ofpectin and the degree of methylation is variable among plant species andmoreover dependent on the age and maturity of the fruit. Among therichest sources of pectins are lemon and orange rind, which can containup to 30% of this polysaccharide.

Pectinases can degrade the carbohydrate polymer either by hydrolysis ofthe α-1,4-glycosidic bond (endo and exopolygalacturonases or bytranselimination reaction (pectin lyases). Pectin esterases candemethylate highly esterified pectin into polygalacturonic acid. Pectinlyases are specific for highly esterified pectins, polygalacturonaseshydrolyse low esterified pectins. Consequently highly esterified pectinscan be degraded by pectin lyases or the combination of pectin esterasesand polygalacturonases (Pilnik 1982).

In the various stages of fruit and vegetable processing pectinases playan important role. Originally pectinases were used for treatment of softfruit to ensure high yields of juice and pigments upon pressing and toclarify raw press juices. Polygalacturonases are used as maceratingenzymes for the production of pulpy nectars, loose cell suspensions thatare the result of limited pectin breakdown particularly in the middlelamella. A combination of several pectinases together with cellulolyticenzymes is needed to almost completely liquefy fruit tissue, therebyfacilitating extraction (Renard et al. 1989). The clarification of applejuices can for example be improved by the combined activity of pectinesterases and polygalacturonases or by pectin lyases for which thehighly esterified apple pectin is an ideal substrate (Ishii andYokotsuka 1973).

Most of the pectinases present in commercial preparations are of fungalorigin. Aspergillus niger is the most important organism for theindustrial production of pectin degrading enzymes. In A. niger thevarious pectinases are not expressed constitutively (Maldonado et al.1989). Pectin or degradation products of the pectin molecule are neededas inducing substances. The fermentation conditions for pectinaseproduction often result in a wide spectrum of pectinases. Moreover, A.niger produces many isoenzymes of the various pectinases. Recentlypatents have been published describing that genes encodingpolygalacturonase (EPO 0421 919, EPO 0 388 593), pectin lyases (EPO 0278 355, EPO 0 353 188) and pectin esterases (EPO 0 388 593) have beenisolated and used for the construction of overproducing transformants.These transformants allow the production of specific enzymes, needede.g. in maceration applications and in studies on the effect of thevarious pectinases in processes like liquefaction and clarification.

Schols et al. (1990b) have described the isolation and characterizationof a cell-wall polysaccharide from apple juice obtained after theliquefaction process in which the juice was released from the apple pulpby the combined action of pectolytic and cellulolytic enzymes. Thesecell-wall polysaccharides resemble the hairy regions of apple pectin (arhamnose-galacturonic acid backbone with arabinose rich side chains) andhave been called Modified Hairy Regions (MHR). Hairy regions are knownto be present not only in apples but also in carrots, grapes andstrawberries and are probably a common part of pectin molecules. Themodified hairy regions are resistant to breakdown by the enzymes presentin most pure and technical pectinase and cellulase preparations. So onlya commercial crude enzyme preparation obtained from Aspergillusaculeatus has been found to be able to depolymerize the rhamnogalacturonbackbone of these fragments. This activity was made visible by measuringthe shift in molecular weight distribution using High Performance GelPermeation Chromatography (HPGPC). Schols et al. (1990a) purified theenzyme responsible for the degradation of the modified hairy regionsprepared from apple juice and called the enzyme rhamnogalacturonase(RGase). The enzyme can split glycosidic linkages in therhamnogalacturonan backbone of (apple) pectins producing, besides othernot yet fully identified reaction products, a range of oligomerscomposed of galacturonic acid, rhamnose and galactose with rhamnose atthe non reducing end, hence the name rhamnogalacturonase (RG-ase) forthis novel enzyme. The oligomers present after incubation of MHR withrhamnogalacturonase were found to be a mixture of a tetramet(Rhamnose(2)-Galacturonic acid(2)) and a hexamer(Rhamnose(2)-Galacturonic acid(2)-Galactose(2) (Colquhoun 1990)). Scholset al (1990a) used various chromatographic steps and column materials toisolate and purify the enzyme with RG-ase activity. Rhamnogalacturonasewas found to be inactive against MHR but was very active towards MHR-Sand MHR-HCl. MHR-S is saponified MHR in which the methoxycarbonyl andacetyl groups have been removed. MHR-HCl is MHR from which the arabinangroups have been removed. Rhamnogalacturonase further exhibited nodegrading activity against a polysaccharide fraction (SPS) present insoy-bean isolates in contrast to the commercial enzyme preparationitself which is known to possess activity against SPS (UK Patent 2 115820). This finding illustrates that the commercial enzyme preparationused is indeed a crude enzyme preparation comprising various enzymeswith different activities. In their paper Schols et al (1990a) suggestthat rhamnogalacturonase may be useful in studies of the structures ofcomplex pectic polysaccharides, but no further applications aresuggested.

No commercial preparations of pure rhamnogalacturonase or comprising adefined and regulated amount of rhamnogalacturonase are presentlyavailable. The only method for obtaining an enzyme with RG-ase activitydescribed sofar is the isolation of the enzyme from the aforementionedcommercial preparation according to Schols et al. 1990a, a method thatis lengthy, requires a large number of steps and is uneconomical.

The object of the present invention therefore is to providerhamnogalacturonase in a process that is economical and can lead to easyproduction of pure forms of the desired enzyme. Furthermore theinvention is directed at novel polypeptides having RG-ase activity andat novel compositions comprising rhamnogalacturonase in a predeterminedamount preferably in an amount greater than 0.01 weight % based on thetotal weight of polypeptides present in said composition, with morepreference for an amount greater than 0.1%. The composition can comprisethe polypeptide having rhamnogalacturonase activity alone or incombination with other (hemi)cellulolytic enzymes or pectinases.

SUMMARY OF THE INVENTION

The present invention is directed at providing recombinant DNA materialcomprising DNA with at least a nucleotide sequence encoding a ripeningform of a polypeptide having rhamnogalacturonase activity.

It is also an object of the present invention to provide a cell capableof expression, preferably capable of overexpression of a ripening formof a polypeptide having rhamnogalacturonase activity encoded byrecombinant DNA material.

The recombinant DNA material comprising a nucleotide sequence encoding aripening form of a polypeptide having rhamnogalacturonase activity cancomprise a nucleotide sequence derivable from an organism that ishomologous to the expression host cell into which cell said nucleotidesequence is incorporated or said nucleotide sequence can be heterologousto the expression host cell.

The expression of the nucleotide sequence encoding a ripening form of apolypeptide having rhamnogalacturonase activity can be regulated byoperably linking said nucleotide sequence to regulatory sequences thatcontrol a gene native to the organism from which said nucleotidesequence has been derived. The regulatory sequences can also be foreigni.e. derived from an organism belonging to a different strain, variety,genus or group of organisms than the organism from which the nucleotidesequence encoding a polypeptide with rhamnogalacturonase activity hasbeen derived. The regulatory regions can be regulatory regions of arhamnogalacturonase gene or regulatory regions of other genes.

Another preferred embodiment of the invention is a cell capable ofoverexpression and secretion of a ripening form of a polypeptide havingrhamnogalacturonase activity, preferably a mature form.

It is yet a further object of the present invention to provide a methodfor the production of a ripening form of a polypeptide havingrhamnogalacturonase activity which may in turn advantageously be used inprocesses requiring degradation and/or modification of pectin, inparticular at processes requiring degradation and/or modification ofplant cell wall material. In particular the invention is directed at aprocess for improved liquefaction of fruit and/or vegetables and at aprocess for preventing and/or removing haze formation in particular thehaze arising after dilution of concentrates derived from fruit and/orvegetables. The invention is also directed at rhamnogalacturonasecomprising products suitable for use in such processes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed at a recombinant DNA materialcomprising a DNA sequence encoding a ripening form of a polypeptidehaving rhamnogalacturonase activity or a precursor of said polypeptideor which recombinant DNA material is capable of producing a ripeningform of a polypeptide having rhamnogalacturonase activity, comprising atleast a part of a nucleotide sequence selected from the group consistingof

a) a nucleotide sequence encoding a ripening form of a polypeptidehaving rhamnogalacturonase activity;

b) a genetic variant of a nucleotide sequence according to a);

c) a nucleotide sequence capable of hybridizing to either of thenucleotide sequences a) or b).

The term "recombinant DNA material" can comprise a DNA molecule, or amixture of various DNA fragments/molecules.

The term "genetic variant" as used herein includes hybrid DNA sequencescomprising at least a part of a nucleotide sequence encoding a ripeningform of a polypeptide having rhamnogalacturonase activity optionallycoupled to regulatory regions such as promoter, secretion and terminatorsignals originating from homologous or heterologous organisms. The term"genetic variant" also includes DNA sequences encoding mutantrhamnogalacturonase polypeptides, i.e. polypeptides comprising mutationsnot affecting the RG-ase activity and degenerate DNA sequences encodingpolypeptides wherein the rhamnogalacturonase activity is retained. Theterm "genetic variant" also includes synthetic DNA sequences encodingpolypeptides having rhamnogalacturonase activity.

The present invention also includes recombinant DNA material comprisingat least a part of a nucleotide sequence capable of hybridizing to atleast a part of the nucleotide sequence encoding a ripening form of apolypeptide having rhamnogalacturonase activity and genetic variantsthereof as described above which may differ in codon sequence due to thedegeneracy of the genetic code or cross species variation. Forhybridisation to occur homology between the DNA material must be higherthan 40%. Preferably the homology is larger than 70%. The hybridisationconditions can be adjusted to correspond to conditions required for thedesired degree of homology. The more stringent the hybridisationconditions, the more homology hybridising sequences will show. A personskilled in the art can carry out such hybridisation tests.

The term "ripening form" refers to any of the different forms in whichan enzyme may occur after expression of the associated gene. More inparticular it refers to both the naturally and not naturally occurringmature form of an enzyme that can result after cleavage of a "leader"peptide and also to any form of an enzyme still comprising a "leader"peptide in any form. In general a "leader peptide" can be a prepropeptide, a pre peptide or a pro peptide.

The recombinant DNA material according to the invention can comprise atleast a part of a nucleotide sequence encoding a ripening form of apolypeptide having rhamnogalacturonase activity wherein said nucleotidesequence can be derived from any organism varying from a mammal to amicroorganism.

With a view to application in processes directed at the production offoodstuffs, a preferred recombinant DNA material according to theinvention will comprise a nucleotide sequence encoding a ripening formof a polypeptide having rhamnogalacturonase activity originating from afoodgrade organism.

As already stated the nucleotide sequence encoding a ripening form of apolypeptide having rhamnogalacturonase activity can be of microbialorigin. Such a sequence can be derived from a microorganism such as afungus preferably a foodgrade fungus. Suitable fungi are the filamentousfungi e.g. the group comprising the genera Aspergillus, Trichoderma,Neurospora, Penicillium and Mucor. Of the genus Aspergillus the speciesof the group comprising Aspergillus niger, Aspergillus awamori,Aspergillus oryzae, Aspergillus sojae, Aspergillus foetidus, Aspergilluscarbonarius, Aspergillus tubigensis, Aspergillus aculeatus andAspergillus japonicus are eminently suitable examples of organisms fromwhich a nucleotide sequence encoding a ripening form of a polypeptidehaving rhamnogalacturonase activity can be derived.

A more concrete preferred embodiment of this aspect of the invention isrecombinant DNA material comprising at least a part of a nucleotidesequence encoding a ripening form of a polypeptide havingrhamnogalacturonase activity with the amino acid sequence shown in FIG.9, (sequence listing no. 7) and even more concretely a recombinant DNAmaterial comprising at least a part of the nucleotide sequence as shownin FIG. 9A-9I (sequence listing no. 7). The genetic variants of thenucleotide sequence of FIG. 9A-9I (sequence listing no. 7) includingsequences encoding mutant rhamnogalacturonase polypeptides anddegenerate nucleotide sequences coding for polypeptides wherein therhamnogalacturonase activity is retained are also part of the invention,as are nucleotide sequences capable of hybridizing to at least a part ofthe nucleotide sequences encoding a polypeptide havingrhamnogalacturonase activity as shown in FIG. 9A-9I (sequence listingno. 7) and genetic variants thereof (as described above), wherein saidnucleotide sequences may differ in codon sequence due to the degeneracyof the genetic code or cross species variation.

A polypeptide having rhamnogalacturonase activity derived fromAspergillus aculeatus was used to obtain the nucleotide sequence andamino acid sequence given in figure 9A-9I (sequence listing no. 7).Based on the cross-reaction observed between antibody raised againstrhamnogalacturonase of Aspergillus aculeatus and enzymes of othermicroorganisms it is clear that various other organisms comprise apolypeptide with rhamnogalacturonase activity.

Rhamnogalacturonase activity has now been found in a number of strainsand derivatives of the genus Aspergillus besides the Aspergillusaculeatus strain used for preparing the commercial preparation (CBS101.43), e.g. Aspergillus niger 402 (CBS 120.49), Aspergillus nigerhennebergii (CBS 117.80), Aspergillus carbonarius (CBS 112.80, CBS420.64), Aspergillus niger nanus (CBS 136.52, CBS 117.48), Aspergillusfoetidus (CBS 121.78, CBS 618.78), Aspergillus tubigensis (CBS 11529),Aspergillus niger intermedius (CBS 559.65) and Aspergillus japonicus(CBS 114.51, CBS 621.78), Aspergillus aculeatus (CBS 115.80, CBS 172.66,CBS 119.49).

The subject invention is therefore directed at polypeptides withrhamnogalacturonase activity derivable from these microorganisms.Furthermore the invention is also directed at antibodies raised againstany such polypeptides with rhamnogalacturonase activity. The inventionis directed at both antibodies with specificity in general forrhamnogalacturonase but also at antibodies specific for one specifictype of rhamnogalacturonase. As illustrated in Example I, a personskilled in the art can arrive at such antibodies in a manner well knownin the art.

The invention is also directed at use of such antibodies capable ofrecognizing at least one antigenic determinant of a polypeptide withrhamnogalacturonase activity for detecting and/or selecting apolypeptide or a cell having rhamnogalacturonase activity in a mannerwell known to a person skilled in the art.

Using hybridisation techniques a part of the isolated DNA encoding apolypeptide having rhamnogalacturonase activity can also be used toscreen other organisms for a DNA sequence having homology with saidisolated DNA. The hybridizing part of the genetic material of the otherorganism can be assumed to comprise at least a part of a nucleotidesequence encoding a ripening form of a polypeptide havingrhamnogalacturonase activity. Using the process for recovering such anucleotide sequence as given in Example I, a person skilled in the artcan derive a nucleotide sequence encoding a ripening form of apolypeptide having rhamnogalacturonase activity from another organism.Use of stringent hybridisation conditions enables selection ofantibodies that bind very strongly to the polypeptide. A person skilledin the art knows what hybridisation conditions to select.

The recombinant DNA material according to the invention can be used toexpress a nucleotide sequence encoding a ripening form of a polypeptidehaving rhamnogalacturonase activity or the recombinant DNA material canbe used as a probe or a primer for detection, isolation or production ofgenetic material encoding at least a part of a ripening form of apolypeptide with rhamnogalacturonase activity or a precursor of suchripening form.

The recombinant DNA material according to the invention can comprise thenucleotide sequence encoding a ripening form of a polypeptide havingrhamnogalacturonase activity operably linked to at least one regulatoryregion capable of directing the expression of said nucleotide sequence.The regulatory regions can be native to the organism from which thenucleotide sequence encoding the polypeptide having rhamnogalacturonaseactivity is derived. Said native regulatory regions can be theregulatory regions that regulate the rhamnogalacturonase gene in theorganism of origin of said polypeptide but can also be regulatoryregions that regulate a different gene in said organism of origin. Aregulatory region other than the native regulatory region that regulatesthe rhamnogalacturonase gene in the organism of origin of said gene willgenerally be selected for its higher efficiency. It is also possible toselect a regulatory region such as a promoter on the basis of otherdesirable characteristics, for example thermo inducibility. Theselection of a desirable regulatory region will be obvious to oneskilled in the art.

In another embodiment the recombinant DNA material according to theinvention can comprise regulatory regions foreign to the organism fromwhich the nucleotide sequence encoding the polypeptide havingrhamnogalacturonase activity is derived operably linked to saidnucleotide sequence. In this instance the regulatory regions can beregulatory regions that regulate a rhamnogalacturonase gene in theforeign organism from which they are derived or can be regulatoryregions that regulate a gene other than the rhamnogalacturonase gene inthe foreign organism.

The selection of a desirable regulatory region will be obvious to oneskilled in the art and will for example depend on the host cell intowhich the recombinant DNA material according to the invention isintroduced. If a heterologous expression host is preferred, meaning thatthe nucleotide sequence encoding a polypeptide havingrhamnogalacturonase activity is derived from another strain of organismthan the host cell (e.g. a different strain, variety, species, genus,family, order, class, division or kingdom) the regulatory region ispreferably a regulatory region derived from an organism similar to orequal to the expression host. For example, if the nucleotide sequence isderived from a fungus and the expression host is a yeast cell, then theregulatory region will be derived from a yeast cell. The regulatoryregion need not however necessarily be derived from the same strain orthe same genus as the host cell, i.c. a yeast cell. The selection of ayeast cell promoter in this instance is required to enable expression ofthe nucleotide sequence.

A regulatory region operably linked to a nucleotide sequence encoding aripening form of a polypeptide having rhamnogalacturonase activity inthe recombinant DNA material according to the invention can be e.g. aconstitutive promoter or an inducible promoter. Especially suited areconstitutive promoters derived from genes encoding enzymes involved inthe glycolytic pathway.

An example of a recombinant DNA material according to the inventioncomprising a strong constitutive fungal promoter operably linked to thenucleotide sequence encoding a ripening form of rhamnogalacturonaseactivity is a recombinant DNA material wherein said promoter is theglyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter. This promoteris preferred for constitutive expression when recombinant DNA materialaccording to the invention is expressed in a fungal expression host.Other examples are pgk, the phosphoglycerate kinase promoter, pki, thepyruvate kinase promoter, TPI, the triose phosphate isomerase promoter,the APC synthetase subunit g (oliC) promoter and the acetamidase (amdS)promoter.

Examples of recombinant DNA material according to the inventioncomprising inducible fungal promoters operably linked to the nucleotidesequence encoding a ripening form of rhamnogalacturonase activity arerecombinant DNA materials, wherein said inducible promoters are selectedfrom the promoters of the following genes: xylanase A (xylA),glucoamylase A (glaA), cellobiohydrolase (cbh), amylase (amy), invertase(suc) and alcohol dehydrogenase alcA, TAKA amylase and amyloglucosidase(AGT). Preferably the inducible xylanase A promoter is selected.

Examples of recombinant DNA material according to the inventioncomprising strong yeast promoters operably linked to the nucleotidesequence encoding a ripening form of rhamnogalacturonase activity arerecombinant DNA materials, wherein said yeast promoters are selectedfrom the promoters of the following genes: alcohol dehydrogenase,lactase, 3-phosphoglycerate kinase, triose phosphate isomerase,α-D-galactose-phosphate uridyl transferase (Ga17) andglyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Examples of recombinant DNA material according to the inventioncomprising bacterial promoters operably linked to the nucleotidesequence encoding a ripening form of rhamnogalacturonase activity arerecombinant DNA materials, wherein said bacterial promoters are selectedfrom the promoters of the following genes: α-amylase, SPO2 andextracellular proteases.

In the same manner that regulating regions foreign to therhamnogalacturonase gene can be coupled to said gene, it is alsopossible to couple a regulating region of a rhamnogalacturonase gene toa heterologous gene. The invention is therefore also directed at anucleotide sequence comprising at least a regulating region of arhamnogalacturonase gene e.g. as indicated in FIG. 9A-9I (sequencelisting no. 7) and at use of such a nucleotide sequence e.g. forregulating the expression of the gene to which said sequence is coupled.

If a heterologous expression host is a yeast or a bacterial strain arecombinant DNA material according to the invention comprising anuninterrupted (intronless) nucleotide sequence encoding a ripening formof a polypeptide having rhamnogalacturonase activity is preferred. Thispreference stems from the fact-that the possibility that theheterologous host does not recognize splicing signals residing on therecombinant DNA material can thus be avoided. Such an uninterruptednucleotide sequence was obtained from a cDNA library constructed fromRNA isolated from cells expressing a nucleotide sequence encoding aripening form of a polypeptide with rhamnogalacturonase activity and ispresented in FIG. 6A-6F, sequence listing no. 5). Alternatively anuninterrupted nucleotide sequence may be obtained by applying one ormore polymerase chain reactions using suitable primers, so as toprecisely remove the introns, using genomic DNA as a template, as isknown to a person skilled in the art.

For the expression in yeast such as Saccharomyces cerevisiae it ispreferable that the introns are removed and that the fungalrhamnogalacturonase leader sequence is replaced by a signal sequencesuitable for yeast such as the signal sequence of the invertase geneensuring correct processing and secretion of the mature polypeptide.

The removal of introns is necessary for expression in bacteria such asBacillus subtilis. In this case for example the α-amylase signalsequence can be used as signal sequence.

A preferred embodiment of recombinant DNA material according to theinvention comprises at least one selection marker. Such a selectionmarker serves to discriminate host cells into which the recombinant DNAmaterial has been introduced from cells that do not comprise saidrecombinant DNA material. This selection marker provided with theappropriate regulatory sequences may reside on the same DNA fragmentcontaining the nucleotide sequence encoding a ripening form of apolypeptide having rhamnogalacturonase activity or can be present on aseparate fragment. In the latter case a co-transformation must beperformed with the various components of the recombinant DNA materialaccording to the invention. The ratio of expression component(containing the nucleotide sequence encoding a ripening form of apolypeptide having rhamnogalacturonase activity)/selection component(with the selection marker) can be adjusted in such a manner that a highpercentage of the selected cells comprising the selection component havealso incorporated the expression component. The term recombinant DNAmaterial as used herein therefore comprises one or more recombinant DNAfragments, wherein the selection marker can be incorporated on the samerecombinant DNA molecule as the nucleotide sequence encoding a ripeningform of a polypeptide having rhamnogalacturonase activity or on adifferent recombinant DNA fragment.

Very often filamentous fungi are transformed through co-transformation.For example a pyrA⁻ strain (pyrA=orotidine-5'-phosphate decarboxylase)can be used as host cell and the recombinant DNA material according tothe invention will comprise a DNA molecule comprising the nucleotidesequence encoding a ripening form of a polypeptide havingrhamnogalacturonase activity and another DNA molecule comprising thepyrA gene. After transformation of the pyrA⁻ strain any resulting pyrA⁺strain will obviously have incorporated some recombinant DNA materialand will most probably also comprise the nucleotide sequence encoding aripening form of a polypeptide having rhamnogalacturonase activity. Veryoften such co-transformation will lead to incorporation of the componentof recombinant DNA material according to the invention comprising thenucleotide sequence encoding a ripening form of a polypeptide havingrhamnogalacturonase activity per host cell in multiple copies (multicopyincorporation). This is a well-known route for producing multicopytranformants in general.

Other well-known selection systems for industrial microorganisms arethose formed by the group of selection markers which do not require amutation in the host organism. Examples of fungal selection markers arethe genes for acetamidase (amdS), ATP-synthetase, subunit 9 (oliC) andbenomyl resistance (benA). Another example of a fungal selection markeris the nitrate reductase system. Exemplary of non-fungal selectionmarkers are the g418 resistance gene (yeast), the ampicillin resistancegene (E. coli) and the neomycin resistance gene (Bacillus), a geneconferring resistance to hygromycin (hph) or a gene conferringresistance to fleomycin (Ble).

Suitable transformation methods and suitable expression vectors providedwith e.g. a suitable transcription promoter, suitable transcriptiontermination signals and suitable marker genes for selecting transformedcells are already known for many organisms including differentbacterial, yeast, fungal and plant species. Reference may be made foryeast for example to Tagima et al. Yeast 1, 67-77, 1985, which showsexpression of a foreign gene under control of the ga17 promoterinducible by galactose in yeast and for Bacillus subtilis for example inEP-A-0,157,441 describing a plasmid pNS48 containing the SPO2 promoteras an expression vector. For the possibilities in these and otherorganisms reference is made to the general literature.

Overexpression of a ripening form of a polypeptide havingrhamnogalacturonase activity may be achieved by the incorporation ofrecombinant DNA material according to the invention in an expressionhost, said recombinant DNA material comprising one or more regulatoryregions (selected for example from promoter and terminator regions)which serve to increase expression levels of the polypeptide of interestfrom said expression host. If desired the polypeptide of interest can besecreted from the expression host. This can be achieved by incorporatingrecombinant DNA material according to the invention as described, saidDNA material further comprising at least one signal sequence (e.g. a preor prepro sequence).

The present invention is not only directed at the recombinant DNAmaterial comprising at least a part of a nucleotide sequence encoding aripening form of a polypeptide having rhamnogalacturonase activity inthe various embodiments as described above but is also directed at acell comprising at least a part of such recombinant DNA material, saidcell being capable of expression of said nucleotide sequence.

Progeny of an expression host comprising recombinant DNA materialaccording to the invention is also embraced by the present invention.

Preferably a cell according to the invention will be capable ofoverexpression of a nucleotide sequence encoding a ripening form of apolypeptide having rhamnogalacturonase activity. Within the context ofthe present invention overexpression is defined as the expression of theripening form of a polypeptide having rhamnogalacturonase activity atlevels above those ordinarily encountered under the same conditions inthe native organism from which said polypeptide originates. In the samecontext overexpression also covers the expression of the ripening formof a polypeptide having rhamnogalacturonase activity in an organismother than the organism from which the nucleotide sequence comprised onthe recombinant DNA material according to the invention can be derived,a so called heterologous organism. The heterologous host organism doesnot normally produce such a ripening form of a polypeptide havingrhamnogalacturonase activity at appreciable levels and the heterologousorganism is therefore only capable of such production after introductionof the recombinant DNA material according to the invention.

As already stated, overexpression of a ripening form of a polypeptidehaving rhamnogalacturonase activity may be achieved by incorporation ofrecombinant DNA material according to the invention.

In order to obtain overexpression recombinant DNA material according tothe invention can be incorporated in a homologous expression host. Theterm "homologous expression host" means that the non transformedexpression host belongs to the same strain or species as the organismfrom which the nucleotide sequence encoding a ripening form of apolypeptide having rhamnogalacturonase activity that is comprised on therecombinant DNA material according to the invention has been derived.

Introduction of the recombinant DNA material according to the inventioninto a homologous expression host will result in the expression hostcomprising at least two nucleotide sequences encoding a ripening form ofpolypeptide having rhamnogalacturonase activity, becoming a so-calledmulticopy transformant.

The overexpression can be further achieved by the introduction of therecombinant DNA material according to the invention into a hostbelonging to a strain other than the strain from which the nucleotidesequence encoding a ripening form of polypeptide havingrhamnogalacturonase activity was isolated a so-called heterologous host,such that the resulting expression host comprises a nucleotide sequenceencoding a ripening form of polypeptide having rhamnogalacturonaseactivity in increased gene copy numbers, becoming a so-called multicopytransformant.

The methods generally known for obtaining multicopy transformants can beused. The recombinant DNA material according to the invention thereforecomprises any embodiment required for obtaining a multicopy transformantcomprising multiple copies of the nucleotide sequence encoding aripening form of polypeptide having rhamnogalacturonase activity.

The overexpression can also be achieved by the introduction of therecombinant DNA material according to the invention in the variousembodiments already described into a host cell such that the host cellcomprises the nucleotide sequence encoding a ripening form ofpolypeptide having rhamnogalacturonase activity under the control of aregulatory region other than the native regulatory region for therhamnogalacturonase gene in the organism from which said nucleotidesequence is derived, said other regulatory region preferably being moreefficient than the native regulatory region. The invention is alsodirected at recombinant DNA material in any of the various embodimentsdescribed further comprising a regulatory region other than the nativeregulatory region for the rhamnogalacturonase gene in the organism fromwhich said nucleotide sequence is derived. Such a host cell can beeither homologous or heterologous. The host cell can comprise one ormore copies of the nucleotide sequence encoding a ripening form ofpolypeptide having rhamnogalacturonase activity comprised on therecombinant DNA material according to the invention.

In some instances it can be preferable to introduce recombinant DNAmaterial according to the invention in such a manner that saidrecombinant DNA material is integrated in the chromosomal DNA of thehost cell. In fungal cells chromosomal integration always takes place insuccessful transformations. No plasmid DNA is maintained. In yeast bothplasmids and integrated DNA can be maintained satisfactorily.

It is possible to introduce recombinant DNA material into the host cellsuch that the genetic properties that are introduced are located onextra-chromosomal DNA most often called "plasmids". Plasmids have theadvantage that they exist normally in the cell in multiple copies whichalso means that a certain gene located on such a plasmid exists in thecell in multicopy form which may result in a higher expression of theproteins encoded by the genes. However, the disadvantage of plasmids isthat they can be unstable resulting in a possible loss of the plasmidsfrom the cells at a certain stage. The loss of a plasmid can beprevented by using a plasmid comprising at least one stretch ofnucleotides capable of hybridizing with chromosomal DNA of thenon-transformed host cell enabling said vector to integrate stably intothe chromosome of said host cell after transformation. Use of a stretchof homologous DNA that is already present in multiple copies in thechromosomal DNA will lead to multicopy insertion of the vector DNAresulting in integrated multimeric DNA comprising one or more copies ofthe nucleotide sequence encoding a ripening form of a polypeptide havingrhamnogalacturonase activity. Another prerequisite for a vectorresulting in DNA integrated in the chromosomal DNA is that the vectordoes not comprise a functional replicon as the vector must be unable tomaintain itself in the host cell unless it is integrated.

The stretch of nucleotides enabling integration is preferably derivablefrom DNA that comprises at least part of a non-essential portion of thechromosome of a non-transformed host cell (in this instance the term"derivable from" implies that the stretch of nucleotides in the vectoraccording to the invention must show enough homology with thechromosomal DNA to enable hybridization for an integration event tooccur). The integration of the vector will subsequently take place insaid non-essential portion of the chromosome of the host cell and willnot lead to the loss of an essential function of the host cell. It ispreferable for the integration to take place in a non-essentialselectable gene of the chromosome of the non-transformed host cell. Thiscan subsequently be a selection criterium for transformed host cells.

In the case of fungal cells it is only possible to successfully obtaintransformants having DNA integrated in the chromosomal fungal DNA asplasmids cannot be maintained in such cells. In fungal cells it is noteven necessary to include homologous chromosomal DNA as multicopyintegration takes place without said homologous DNA. In the case ofyeast cells it is optional to have the desired DNA in the transformanteither as a plasmid or as integrated DNA. For integration in yeast cellsDNA sequences homologous to chromosomal DNA must be present.

A preferred embodiment of the invention is directed at a cell comprisingrecombinant DNA material according to the invention in any of theembodiments described, wherein said cell is capable of secreting aripening form in particular capable of secreting a mature form of apolypeptide with rhamnogalacturonan activity as encoded by saidrecombinant DNA material. It is often desirable for the ripening form ofa polypeptide having rhamnogalacturonase activity to be secreted fromthe expression host into the culture medium as said polypeptide may bemore easily recovered from the medium than from the cell. Preferably themature form of the rhamnogalacturonase will be secreted into the culturemedium.

The term "secretion" in the subject invention comprises the polypeptidecrossing a cell wall or a cell membrane. The polypeptide can pass such acell wall or membrane into the culture medium but can also remainattached to said cell wall or cell membrane. The polypeptide can alsopass a cell membrane into the periplasmic space and not into the culturemedium. The processing c.q. secretion route to be followed by theripening form of a polypeptide having rhamnogalacturonase activity willdepend on the selected host cell and the composition of the recombinantDNA material according to the invention. Most preferably, however, thepolypeptide will be secreted into the culture medium.

The cell according to the invention can comprise recombinant DNAmaterial in any of the various embodiments described further comprisingDNA encoding the native leader sequence (pre or prepro) of thepolypeptide having rhamnogalacturonase activity. In another embodimentthe cell according to the invention can comprise recombinant DNAmaterial further comprising DNA encoding for foreign leader sequences(pre or prepro) instead of the native leader sequences. The invention isalso directed at recombinant DNA material comprising DNA encoding themature polypeptide having rhamnogalacturonase activity coupled to DNAencoding a leader sequence foreign to the polypeptide havingrhamnogalacturonase activity.

An increase in the expression of a polypeptide havingrhamnogalacturonase activity can result in the production of polypeptidelevels beyond those the expression host is capable of processing andsecreting resulting in a build up of polypeptide product within the hostcell creating a bottle neck in the transport of the polypeptide throughthe cell membrane or cell wall. Accordingly the present invention isalso directed at a cell comprising recombinant DNA material in any ofthe various embodiments described comprising heterologous signalsequences to provide for the most efficient secretion of therhamnogalacturonase from the chosen expression host and the invention isalso directed at said recombinant DNA material.

A heterologous secretion signal sequence may be chosen such that it isderived from the same strain as the organism from which the otherregulatory regions of the nucleotide sequence encoding a ripening formof a polypeptide having rhamnogalacturonase activity have been derived,preferably from the same gene. For example the signal of the highlysecreted amyloglucosidase protein may be used in combination with theamyloglucosidase promoter itself as well as in combination with otherpromoters.

Examples of preferred heterologous secretion signal sequences are thoseoriginating from the glucoamylase A or xylanase A gene for fungi, theinvertase gene for yeast and the α-amylase gene for Bacillus.

Hybrid secretion sequences may also advantageously be used within thecontext of the present invention.

In general terminators of transcription are not considered to becritical elements for the overexpression of genes. If desired, aterminator of transcription may be selected from the same gene as thepromoter or alternatively the homologous terminator may be employed. Infact any terminator can be employed.

Factors such as size (molecular weight) the possible need forglycosylation or the desirability of the secretion over the cellmembrane or cell wall or into the medium of the rhamnogalacturonase playan important role in the selection of the expression host.

Partly depending on the selected host cell the nucleotide sequenceencoding a polypeptide having rhamnogalacturonase activity will be usedeither with or without introns occurring in said DNA sequence eitherwith its own promoter and/or transcription termination signals ororiginating from another gene and either with its own leader sequence orwith a signal sequence originating from another gene.

In principle the invention knows no special limitations with respect tothe nature of the cells comprising recombinant DNA material according tothe invention. Cells according to the invention may be important asagents for multiplying the recombinant DNA material or as agents forproducing a ripening form of a polypeptide having rhamnogalacturonaseactivity.

Those expression hosts capable of overexpression of a nucleotidesequence encoding a ripening form of a polypeptide havingrhamnogalacturonase activity are preferred. In particular an expressionhost cell capable of secretion of a ripening form of polypeptide havingrhamnogalacturonase activity is preferred.

The expression hosts can be selected from the group consisting ofbacterial cells, fungal cells, yeast cells and plant cells, with apreference for foodgrade host cells.

Preferred examples of eminently suited host cells are

a) fungal cells, in particular filamentous fungal cells, such as afungal cell from the group comprising the genera Aspergillus,Trichoderma, Neurospora, Penicillium and Mucor. Examples of particularspecies that are suitable as host cell are fungal cells of one of thespecies Aspergillus niger, Aspergillus awamori, Aspergillus oryzae,Aspergillus nidulans, Aspergillus sojae, Aspergillus tubigensis,Aspergillus aculeatus, Aspergillus foetidus, Aspergillus carbonarius,Aspergillus japonicus, Trichoderma reesei and Trichoderma viride;

b) yeast cells, for example of the genera Saccharomyces, Kluyveromyces,Hansenula and Pichia, in particular yeast cells of one of the speciesSaccharomyces cerevisiae, Saccharomyces carlbergensis, Kluyveromyceslactis, Kluyveromyces marxianus, Hansenula polymorpha and Pichiapastoris;

c) plant cells of a plant genus selected for example from the groupconsisting of wheat, barley, oats, maize, pea, potato, apple, grape,chicory, coffee, tea and tobacco such as plant cells of one of thespecies Solanum tuberosum and Nicotiana tobaccum; and

d) bacterial cells, preferably gram positive bacterial cells, forexample of one of the bacterial genera Bacillus, Lactobacillus andStreptococcus such as bacteria of the species Bacillus subtilis orBacillus licheniformis.

The host cell to be selected for recombinant DNA material according tothe invention will amongst others depend on the application for whichthe resulting polypeptide having rhamnogalacturonase activity isdestined.

A preferred cell according to the invention is a foodgrade cell. Thispreference stems from the fact that products of such foodgrade cells canbe used in processes for producing foodstuffs. Bacteria from the genusBacillus are very suitable as expression host cells because of theircapability to secrete proteins into the culture medium. Alternatively ahost selected from the group of yeasts or fungi may be preferred. Insome instances yeast cells are easier to manipulate than fungal cells.However, some proteins are either poorly secreted from the yeast cell orin some cases are not processed properly (e.g. hyperglycosylation inyeast). In these and other instances a fungal host organism can beselected. A fungal host is often suitable if it has GRAS status(GRAS=generally regarded as safe). In general, eukaryotic hosts havebeen found to have a high productivity of secreted active polypeptides.In fact fungal hosts are very often used in industrial processes,particularly suitable examples of a host cell are therefore Aspergillusniger and Aspergillus niger var. awamori. These particular species ofAspergillus have previously been demonstrated to be excellent host cellsfor industrially producing enzymes. A person skilled in the art is ableto obtain multicopy transformants of these species.

In the case of polypeptide production it is possible to use theexpression host cell to produce polypeptide and to subsequently eitherisolate the polypeptide from the culture medium or use the mediumcontaining the polypeptide as such after removal of the cells. It iseven possible to use the cells themselves to produce the polypeptide insitu in the process for which the polypeptide having rhamnogalacturonaseactivity is required. In the preparation of foodstuffs such a hoststrain that is to be used directly can only be used if it is a foodgrade host strain.

If the polypeptide is required in extremely purified forms or ifparticular contaminants are deleterious to the application of theresulting polypeptide, the expression host cell can be selected to avoidsuch problems. The presence of protease as contaminant for example isnot desirable. Presence of protease, in particular, should be avoidedwhen long term storage is being contemplated. It is possible to use sizeexclusion chromatography involving BioGel P100 (BioRad) to effectivelyreduce the content of undesirable proteases by 80-90%. In addition tosize exclusion chromatography, protease activity can be removed by otherwell-known techniques such as ion exchange chromatography, bentonitetreatment, or pH/temperature inactivation. In order to avoid such costlyand complicated steps it is however preferable to select a proteasenegative strain as host cell.

The subject invention is also directed at a ripening form of apolypeptide with rhamnogalacturonase activity wherein said ripening formis obtainable by expression of the recombinant DNA material according tothe invention. In particular a microorganism belonging to the genusAspergillus is a suitable source of a ripening form of a polypeptidewith rhamnogalacturonase activity according to the invention. Theinvention is preferably directed at a mature form of a polypeptide withrhamnogalacturonase activity as no further treatment of said polypeptideis necessary before using said polypeptide in a desired process. Inparticular the invention is directed at a ripening form of a polypeptideas encoded by a part of the nucleotid sequence of FIG. 9 (sequencelisting no. 7). A ripening form of a polypeptide havingrhamnogalacturonase activity, said ripening form being encoded by a partof any equivalent nucleotide sequence encoding a polypeptide with anequivalent tertiary structure having rhamnogalacturonase activity alsoforms part of the invention.

The invention is also directed at a process for producing a ripeningform of a polypeptide having rhamnogalacturonase activity comprisingculturing a transformed cell previously described in the specificationunder such conditions that said cell is capable of expressing DNAmaterial capable of producing a ripening form of a polypeptide withrhamnogalacturonase activity and optionally isolating the resultingripening form of the polypeptide having rhamnogalacturonase activity.The expression of the polypeptide with rhamnogalacturonase activity canbe effected by culturing expression host cells that have beentransformed with the recombinant DNA material comprising a nucleotidesequence encoding a ripening form of a polypeptide havingrhamnogalacturonase activity in a conventional nutrient fermentationmedium.

The invention is also directed at a composition comprising a ripeningform of a polypeptide having rhamnogalacturonase activity obtainablethrough expression of recombinant DNA material as disclosed herein or bya process as disclosed herein. Such a composition, which is suitablefor:

improving the extraction of soluble solids from vegetable material orfor

improving the functionality of pectin-containing vegetable material orfood material or for

the degradation or modification of pectin or pectin-containing vegetablematerial or plant cell wall material may comprise 0.01-100, preferably0.1-100 weight % of polypeptide having rhamnogalacturonase activityaccording to the invention, based on the total weight of polypeptide inthe composition.

The invention is also directed at a composition comprising viable, deador lysed cells comprising a recombinant DNA sequence encoding a ripeningform of a polypeptide having rhamnogalacturonase activity as disclosedherein or such cells in any other form or extracts of such cells. Othercomponents capable of the degradation or modification of pectin orpectin-containing vegetable or plant cell wall material may beincorporated in the compositions as disclosed above.

The fermentation medium can comprise an ordinary culture mediumcontaining a carbon source, a nitrogen source, an organic nitrogensource and inorganic nutrient sources. The medium can also containinducing compounds which activate the expression of the nucleotidesequence encoding a polypeptide having rhamnogalacturonase activity. Theselection of the appropriate medium may be based on the choice ofexpression host and/or based on the regulatory requirements of therecombinant DNA material. Such media are well-known to those skilled inthe art. The medium may, if desired, contain additional componentsfavouring the transformed expression host over other potentiallycontaminating microorganisms. In the-case of production of thepolypeptide having rhamnogalacturonase activity for food processing suchadditional components are necessarily also food grade.

After fermentation the cells can be removed from the fermentation brothby means of centrifugation or filtration. Depending on whether the hostcell has secreted the polypeptide having rhamnogalacturonase activityinto the medium or whether said polypeptide is still connected to thehost cell in some way either in the cytoplasm, in the periplasmic spaceor attached to or in the membrane or cell wall, the cells can undergofurther treatment to obtain the polypeptide.

In the latter case, where the polypeptide is still connected to the cellin some manner, recovery of the polypeptide can for example beaccomplished by rupturing the cells for example by high pressuredisruption, sonication, enzymatic digestion or simply by cell autolysisfollowed by subsequent isolation of the desired product. The polypeptidecan be separated from the cell mass by various means. In one such methodthe cells are disrupted by the protease ficin and subjected toultrafiltration. The polypeptide is subsequently precipitated with anorganic solvent such as methanol or acetone. Such isolation methods arewell known to a person skilled in the art.

The polypeptide isolated from microbial cells is generally purified byconventional precipitation and chromatographic methods. Such methodsinclude amongst others methanol, ethanol, acetone and ammonium sulfateprecipitation and ion exchange and hydroxy apatite chromatography. Suchpurification methods are well known to a person skilled in the art.

The compositions as disclosed above comprising a polypeptide withrhamnogalacturonase activity may be used in a process requiring theextraction of components from vegetable material or for improving thefunctionality of pectin or pectin-containing vegetable material, foodmaterial or plant cell wall material. Improving the functionality inthis respect comprises modifying the properties of vegetable or foodmaterial or plant cell wall material in such a way that it may increasethe usefulness of the vegetable or food material or plant cell wallmaterial. This increased usefulness may be expressed by a loweredsensitivity to the quality of the raw materials when processingvegetable or plant cell wall material or it may be expressed by animprovement in processing, enhanced productivity etcetera. In thisrespect, the functionality of products like tea leaves, coffee (beans),flour, dough etcetera may be improved.

The invention is further directed at processes requiring degradationand/or modification of pectin, in particular at processes requiringdegradation and/or modification of pectin-containing vegetable or plantcell wall material, in which processes a polypeptide havingrhamnogalacturonase activity or a cell capable of expressing such apolypeptide as described above is used.

Rhamnogalacturonase can be an important enzyme in various applications.In addition to the present technical pectolytic and cellulolytic enzymesrhamnogalacturonase can improve liquefaction to the extent that a stateof almost complete liquefaction of fruit pulp, resulting in higherextraction yields, can be attained. Rhamnogalacturonase opens thepossibility of juice production from tropical fruits, at present a verydifficult technology. The invention is therefore in particular directedat industrial processes, such as the liquefaction and/or maceration offruit pulp and the extraction of juices from plant material. Theinvention is directed at such processes using any vegetables or fruitsuch as carrots, apples, grapes, strawberries, tropical fruits andchicory. Furthermore as the interaction between arabinans is consideredto be the cause of undesirable haze formation in concentrated fruit andvegetable juices, in particular in apple juice, the use ofrhamnogalacturonase alone or in combination with arabinase can preventhaze formation in concentrated fruit and/or vegetable juices as well asin coffee and tea or in fact in any beverages comprising plant material.The use of rhamnogalacturonase alone or in combination with arabinasecan also prevent haze formation in brewing processes.

Rhamnogalacturonase alone or in combination with other pectinases and(hemi)cellulolytic enzymes e.g. arabinases can result in new macerationproducts, which can be used especially in a process for the productionof nutritional food preparations or ingredients for foodstuffs, e.g.baby food.

Also the quality of fibrous material containing Rha-GalA poly-(gums) doligosaccharides (such as present in beet pulp and potato fibers) can beimproved by use of rhamnogalacturonase.

Since pectin is an essential part of the plant cell-wall tissue theextraction of oils, gums, natural colours, flavours or flavourprecursors from plant biomass can also be facilitated usingrhamnogalacturonase optionally in combination with other pectinases and(hemi)cellulolytic enzymes.

The invention is also directed at any products derived from theabovementioned processes. Such products comprise beverages derived fromplant material, e.g. fruit juice, coffee, tea, beer, wine, cider andnutritional preparations and ingredients for foodstuffs, e.g. for babyfood and new maceration and/or liquefaction products.

BRIEF DESCRIPTION OF TABLES AND FIGURES

Table 1: Viscosity reduction in apple hot mash experiments. 1. Additionof Biopectinase LQ at 500 g/tonne. 2. As 1+addition ofrhamnogalacturonase containing fermentation broth from A. aculeatus CBS115.80

Table 2: Volume (ml), Brix, % Yield at 10.4 Brix, pH and pectin test ofa centrifuged 30 g apple hot mash sample 2 hours after incubationwith 1. Biopectinase LQ at 500 g/tonne. 2. As 1+addition ofrhamnogalacturonase containing fermentation broth from A. aculeatus CBS115.80. 3. No enzymes added.

FIG. 1: Dionex BioLC/HPAE chromatogram of isolated Modified HairyRegions without addition of enzymes (A,B), after incubation withBiopectinase (C,D) and after incubation withBiopectinase+rhamnogalacturonase containing fermentation broth fromAspergillus aculeatus (E,F). Entire chromatogram shown left, detail of20-35 minutes retention time shown right.

FIG. 2: Elution profile of HPLC separation of rhamnogalacturonase CNBrfragments from Aspergillus aculeatus.

FIG. 3: Partial amino-acid sequence of fragments of Aspergillusaculeatus rhamnogalacturonase generated by cleavage with cyanogenbromide (CNBr fragment #7 and #15) and after cleavage withEndoproteinase Lys-C (Elys fragment #7 and #5).

FIG. 4: Western blot analysis, using rhamnogalacturonase antibodies, ofsupernatant of Aspergillus aculeatus fermentation broth at several hoursafter a switch (at t=0) from sucrose to apple pectin. Lanes indicatehours after transfer.

FIG. 5: Map of plasmid pUR7510, containing a cDNA fragment comprisingthe entire coding region of the Aspergillus aculeatusrhamnogalacturonase gene (rhgA).

FIGS. 6A-6F: Nucleotide sequence of a cDNA copy of the Aspergillusaculeatus rhamnogalacturonase gene (rhgA) and amino acid sequencederived therefrom.

FIG. 7: Restriction map of the genomic DNA of Aspergillus aculeatus inthe region of the rhamnogalacturonase gene.

FIG. 8: Map of plasmid pUR7511, containing a genomic DNA fragmentcomprising the Aspergillus aculeatus rhamnogalacturonase gene.

FIGS. 9A-9I: Complete nucleotide sequence of the Aspergillus aculeatusrhamnogalacturonase gene (rhgA), including flanking regions, and aminoacid sequence derived therefrom.

FIG. 10: Restriction map of the genomic DNA of Aspergillus niger in theregion of A) rhgl (4.5 kb BamHI fragment) and B) rhgII (4.5 kb EcoRIfragment).

FIG. 11: Southern analysis of Aspergillus niger and Aspergillusaculeatus genomic DNA after digestion with BamHI and EcoRl with the 2.5kb BamHI- HindIII fragment of rhgl from Aspergillus niger as probe. A.aculeatus genomic DNA lane 1 (BamHI digestion) and lane 2 (EcoRldigestion). A. niger genomic DNA lane 3 (BamHI digestion) and lane 4(EcoRl digestion). Lane 5: Marker.

FIG. 12: Western blot analysis, using rhamnogalacturonase antibodies, ofsupernatant of Aspergillus aculeatus transformants 75 (lane 2, lane 3 (5times diluted) and lane 4 (25 times diluted) and transformant 67 (lane5, lane 6 (5 times dilution) and lane 7 (25 times dilution) and wildtype Aspergillus aculeatus 115.80. (lane 1,8)

FIG. 13: Map of plasmid pUR2930, comprising the promoter region of theAspergillus niger var. awamori xylA gene.

FIG. 14: Schematic representation of the construction pathway for aplasmid comprising the rhgA gene of Aspergillus aculeatus under thecontrol of the xylA promoter of Aspergillus niger var. awamori by meansof PCR.

FIG. 15: Western blot analysis, using rhamnogalacturonase antibodies, ofsupernatant of 6 transformants of Aspergillus niger var. awamoricontaining the A. aculeatus rhamnogalacturonase gene under the controlof the A. niger var. awamori xylA promoter (lane 1 until 6) and A.aculeatus wild type (wt) supernatans induced on apple pectin.

FIG. 16: Dionex BioLC/HPAE chromatogram of isolated Modified HairyRegions without addition of enzymes (A,B) and after incubation withrhamnogalacturonase containing fermentation broth from Aspergillus nigervar. awamori containing the A. aculeatus rhamnogalacturonase gene underthe control of the A. niger var. awamori xylA promoter (B). Entirechromatogram shown left, detail of 20-35 minutes retention time shownright.

FIG. 17: Map of plasmid pUR2904, an Escherischia coli--Saccharomycescerevisiae shuttle vector.

FIG. 18: Map of a plasmid (pUR7513), comprising the Saccharomycescerevisiae invertase signal sequence and the rhgA gene of Aspergillusaculeatus under the control of the ga17 promoter of Saccharomycescerevisiae.

EXAMPLE 1

Use of rhamnogalacturonase from Aspergillus aculeatus in apple juicemanufacturing

1.1. Production of rhamnogalacturonase by Asperillus aculeatus

1.1.1. Isolation of rhamnogalacturonase and preparation of antibodies

Rhamnogalacturonase was isolated using the method and the commercialpreparation described by Schols et al. (1990a). The purifiedrhamnogalacturonase was used for the immunisation of two mice using themethod described by van der Veen et al. (1991). The antibodies obtainedwere used for screening and purification of rhamnogalacturonase fromAspergillus aculeatus.

1.1.2. Identification of strains of filamentous fungi producingrhamnogalacturonase

Various strains of different species of filamentous fungi were culturedin 50 ml conical shake flasks in order to compare their naturalproduction levels of rhamnogalacturonase. The working volume was 30 ml,containing a medium of the following composition:10 gram sugar beet pulpin 1 liter minimal medium containing 6 gram NaNO₃, 0.5 gram KCl, 1.5gram KH₂ PO₄, 0.9 ml 10 N KOH, 0.5 gram MgSO₂.7 H₂ O and 1 ml of a1000×concentrated trace element solution according to Visniac and Santer(1957). Shake flasks were incubated with spores (10E6/ml) and incubatedfor 48 hours at 30° C. onase gene under the control of the A. niger var.awamori xylA promoter (B). Entire chromatogram shown leftfiltrate wasanalysed by SDS PAGE gel electrophoresis followed by Western blotting(Burnette 1981). Loading buffer (4 times loading buffer contains 1.25 MTris/HCl pH 6.8, 10% sodium dodecyl sulphate, 20% 2-mercapto ethanol,50% glycerol and 50 ug/ml bromophenol blue) was added to the culturefiltrate and the samples were boiled for 5 minutes prior to loading ontothe gel. Western blots were analysed for cross-reactivity withantibodies raised against purified rhamnogalacturonase. Various strainsof Aspergillus showed 1 distinct band after hybridisation withrhamnogalacturonase antibodies, with molecular weights ranging from51000 D-63000 D. The following strains were able to secrete a proteinshowing cross reactivity with rhamnogalacturonase antibodies:Aspergillus japonicus (CBS 114.51, CBS 621.78), Aspergillus aculeatus(CBS 115.80, CBS 172.66, CBS 119.49), Aspergillus niger 402 (CBS120.49), Aspergillus niger hennebergi (CBS 117.80), Aspergillus nigerintermedius (CBS 559.65), Aspergillus niger nanus (CBS 136.52, CBS117.48), Aspergillus foetidus (CBS 121.78, CBS 618.78), Aspergilluscarbonarius (CBS 112.80, CBS 420.64). Consequently the antibodies raisedagainst rhamnogalacturonase can be used to screen microorganisms for thesynthesis of rhamnogalacturonase. The organisms described above containa gene encoding a protein which shows cross reactivity with antibodiesraised against rhamnogalacturonase and therefore can be used to isolatesuch a gene and, in principle, can be used for the overproduction ofrhamnogalacturonase.

1.1.3. Rhamnogalacturonase production by Aspergillus aculeatus CBS115.80 and CBS 172.66 on several substrates.

Since the absolute amount of rhamnogalacturonase produced under theconditions described above was rather low, Aspergillus aculeatus CBS115.80 and CBS 172.66 were used in shake flasks experiments in order tocompare the production of rhamnogalacturonase on several substrates.Strains were grown as described above on minimal medium and 5 g/l Difcoyeast extract, 2 g/l casamino acids and 30 g/l sucrose. After growth for24 hours cells were harvested by centrifugation and resuspended inminimal medium and10 gram per liter of the tested substrate. Crossreactivity with antibodies raised against rhamnogalacturonase was foundafter transfer to rhamnose (20 gram/liter) in combination withgalacturonic acid (20 gram per liter) and on apple pectin, citruspectin, beet pectin and sugar beet pulp. No cross reactivity withantibodies raised against rhamnogalacturonase was found after transferto media containing simple carbon sources such as sucrose, glucose,fructose, rhamnose or galacturonic acid. Transfer to lower ratios ofrhamnose/galacturonic acid (1:2, 1:5 and 1:10, based on 20 gram perliter galacturonic acid) also resulted in rhamnogalacturonaseproduction. It is clear that rhamnogalacturonase is not producedconstitutively but that growth conditions determine the expression ofthe rhamnogalacturonase gene.

1.1.4. Fermentative production of rhamnogalacturonase Aspergillusaculeatus CBS 115.80 and CBS 172.66

Aspergillus aculeatus CBS 115.80 and CBS 172.66 were cultured inChemoferm glass 10 liter fermenters equipped with a magnetically driveneight blade impeller. The dissolved oxygen tension was measured with anIngold oxygen probe, the pH was determined with an Ingold pH electrodeand the temperature was measured using a PT100 sensor. The workingvolume of the fermenter was 8 liter. Applikon ADI 1020 control unitswere used for control of pH, temperature, pO2, gass inlet (3 l/min) andstirrer speed (600-1000 rpm). During the fermentation the pH was kept at4.5 by the addition of a 12.5% ammonia solution and the temperature waskept at 30° C. Fermenters were inoculated with 300 ml of a germinatedspore suspension (final concentration 10E6/ml). Spores were germinatedfor 6 hours at 30° C. in a shaking incubator in the minimal mediumdescribed above.

Aspergillus aculeatus CBS 172.66 and CBS 115.80 were cultured accordingto the method described on minimal medium with10 gram/liter sucrose ascarbon source. After growth of the fungus by consumption of sucrose afeed of rhamnose (0.2 gram/liter/hour) and galacturonic acid (0.4gram/liter/hour) was connected. Rhamnogalacturonase was detected 3 hoursafter connection of the feed, as judged by Western analysis of thesupernatant with the rhamnogalacturonase antiserum. Two days after startof the fed-batch the fermentations were stopped and fermentation broth,obtained after filtration of the biomass, was concentrated 10 timesusing an asahi hollow fiber kidney. The concentrated fermentation brothwas again analysed for the presence of rhamnogalacturonase by Westernblot analysis and used in activity tests on modified hairy regions.

In another fermentation experiment Aspergillus aculeatus CBS 115.80 wascultured according to the method described on minimal medium containing5 grams/liter sucrose, 5 grams/liter apple pectin and 5 grams/litercitrus pectin. The fermentation process was carried out for 72 hours,followed by filtration of the biomass and concentration of thefermentation liquid as described above. The concentrated fermentationbroth contained rhamnogalacturonase, as judged by Western blot analysiswith the rhamnogalacturonase antibodies and was used in activity testson modified hairy region and in apple hot mash application trials.

1.2. Activity of rhamnogalacturonase from Aspergillus aculeatus in applejuice manufacturing

1.2.1. Activity of rhamnogalacturonase from Aspergillus aculeatus onmodified hairy regions

For the isolation of modified hairy regions, Golden Delicious apples (10kg) were crushed in a Magimix Cuisine Systeme 3000 and treated with anenzyme preparation (Biopectinase 200 L 0.05%) from Quest Internationalfor 4 hours at 55° C. After centrifugation (Sorvall RC-5B) at 8000 g for30 minutes the supernatant was ultrafiltered and concentrated in aPellicon microfiltration unit having a molecular weight cut off of50.000. The residue was dialysed and lyophilized.

The isolated polysaccharide was characterised by analyzing enzymaticdegradation products. 5 ml of 0.2% solutions of the isolated ModifiedHairy Regions in 0.05 M sodium acetate buffer (pH=5.0) were incubatedwith 10 μl enzyme preparations for 2 hours at 50° C. Analysis of theformed products was performed on a Dionex BioLC/HPAE chromatographysystem or by measuring the increase in the reducing end-groups makinguse of the DNS method. The Dionex system uses a Carbo Pac PA-1 anionexchange column (25 cm, 4 mmi.d.) and a CarboPac PA-1 Guard. The columnwas loaded with 25 μl of the solution (0.2%) and eluted with a lineargradient of 0-0.5 M NaOAc in 0.1 N NaOH during 50 minutes. The flow ratewas 1.0 ml/min and the process was monitored using a PE detector.

For the reducing end group method the DNS reagent was prepared asfollows: 20.0 grams of 2-hydroxy-3,5 dinitrobenzoic acid (Merck 800141)was suspended in 400 ml distilled water. With continuous magneticstirring, 300 ml of NaOH solution (32 grams in 300 ml distilled water)was gradually added to this suspension. The solution was warmedcautiously to 45° C. until it was clear. Rochelle salt (600 g,K-Na-tartrate, Merck 8087) was added under continuous stirring. Thesolution was diluted to 2000 ml with distilled water and stored in adark bottle at room temperature. Using the DNS method, 0.5 ml of thereaction mixture (MHR and enzyme preparation after 2 hours of incubationas described above), was added to 1.5 ml demi-water and 2 mlDNS-solution. The solution was boiled for 10 minutes and after coolingto room temperature the extinction was measured at 543 nm.

FIG. 1 shows the Dionex pattern of isolated Modified Hairy Regionswithout the addition of enzymes (FIG. 1a), incubated with BiopectinaseOS (1C), and incubated with 10 μl rhamnogalacturonase containingfermentation broth from Aspergillus aculeatus CBS 115.80 cultured on acombination of sucrose, apple pectin and citrus pectin as describedabove (1E). It is clear that the isolated Modified Hairy Regions canonly be degraded by rhamnogalacturonase containing fermentation brothand not by Biopectinase (containing other pectinases e.g.polygalacturonases, pectinesterases and petin transeliminases).

Using the DNS method the reducing sugars obtained after incubation ofthe modified hairy regions with 10 μl rhamnogalacturonase containingfermentation broth from Aspergillus aculeatus CBS 115.80 cultured on acombination of sucrose, apple pectin and citrus pectin as describedabove resulted in an extinction of 1.06 at 543 nm. Addition ofrhamnogalacturonase containing fermentation broth after fed batchcultivation on rhamnose and galacturonic acid as described aboveresulted in a extinction of 0.52 at 543. Biopectinase was used in thismethod as a control resulting in extinctions of 0.32 at 543 nm.

Modification of the isolated Modified Hairy Regions with endoarabinaseor with arabinofuranosidase did not result in futher degradationmonitored either by the DNS method or by the Dionex system. Consequentlyrhamnogalacturonase containing fermentation broth from Aspergillusaculeatus CBS 115.80 showed good activity in the degradation of modifiedhairy regions due to the activity of rhamnogalacturonase. The highestactivity was found after cultivation on pectin based media.

1.2.2. Use of rhamnogalacturonase from Aspergillus aculeatus in applejuice manufacturing

Apples were peeled, chopped, depipped and mashed to a fine puree. Applemash was distributed into 500 g aliquots and preincubated at 55° C. Themashes were treated with enzyme preparation as shown below and incubatedfor 2 hours at 55° C. Viscosity of mash was measured several times usinga Brookfield viscometer with Helipath stand attachment and T barspindle. After 2 hours of incubation a 30 g sample was removed from eachmash and centrifuged for 20 minutes at 10.000 rpm. The volume, pH andBrix of the juice were measured. The pectin level of the juice wasassessed by a standard alcohol test (1 part juice/2 parts isopropanol).Concentrated fermentation broths were tested in the presence ofBiopectinase LQ, containing cellulases and various pectinases. Theresults of a typical experiment in which 3 ml of a concentratedrhamnogalacturonase containing fermentation broth from a sucrose/pectinfermentation of Aspergillus aculeatus CBS 115.80 was evaluated is shownin Table 1 and 2. It is clear that the addition of therhamnogalacturonase containing broth resulted in an improved viscosityreduction of the mash especially in the early stages of incubation. Alsoa significant increase in juice brix and juice yield was observed.Inclusion of rhamnogalacturonase containing fermentation broth resultedin a lowering of juice pH and a clearer juice containing less pectinmaterial. Considering the observed activity of rhamnogalacturonase fromAspergillus aculeatus on modified hairy regions and the performance inapplication trials this species was used as starting material for theisolation of the rhamnogalacturonase gene.

                  TABLE 1                                                         ______________________________________                                        Time course of viscosity reduction (cps) in apple                             hot mash experiments: Effect of rhamnogalacturonase in                        addition to Biopectinase LQ. T = time in minutes                                     T = 0  T = 15  T = 30   T = 60                                                                              T = 120                                  ______________________________________                                        Biopectinase                                                                           55692    17745   8034   7098  5518                                   Biopectinase                                                                           55692    11407   6942   5440  5525                                   rhamnogalac-                                                                  turonase                                                                      ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Volume, brix, yield, pH and cloudiness after                                  centrifugation of 30 gram apple hot mash, incubated for 2                     hours with various enzymes.                                                                         % yield                                                        volume         at 10.4         pectin                                         (ml)    Brix   brix       pH   test                                    ______________________________________                                        Biopectinase                                                                           24        11.3   86.9     3.66 hazy                                  Biopectinase                                                                           25        11.8   94.5     3.60 clear                                 rhamnogalac-                                                                  turonase                                                                      No enzymes                                                                             23        10.4   76.6     4.05 gel                                   added                                                                         ______________________________________                                    

EXAMPLE 2

Cloning and analysis of the Aspergillus aculeatus rhgA gene

2.1. Identification of partial amino acid sequences of A. aculeatusrhamnogalacturonase

2.1.1. Isolation of rhamnogalacturonase from Aspergillus aculeatus172.66

Aspergillus aculeatus CBS 172.66 was cultured in a 10 liter fermenteraccording to the method described above on minimal medium supplementedwith 5 grams per liter peptone and 10 grams per liter sugar beet pulp.After 50 hours of cultivation cells were removed by filtration throughmiracloth filters and the culture filtrate was used for the purificationof rhamnogalacturonase. Culture filtrate was diluted 5 times with waterand brought to pH 6.5 with NaOH after which DEAE Sephadex-A50 (20 g/l),equilibrated in 20 mM sodium phosphate buffer pH 6.5, was added. Thesuspension was mixed for two hours and the DEAE Sephadex-A50 wascollected on a glass funnel. The bound protein was eluted in steps with0.5 M NaCl and 20 mM sodium phosphate buffer pH 6.5 and was collected infractions. The rhamnogalacturonase containing fractions were identifiedby SDS-PAGE followed by Western blotting using the rhamnogalacturonaseantibodies described above. The rhamnogalacturonase containing fractionswere dialysed against distilled water and loaded on a DEAE SepharoseFast Flow column (dimensions 2.6×9 cm) equilibrated with 20 mMpiperazine/HCl buffer pH 6.0 and eluted with a 1000 ml NaCl gradient(0-0.5 M) in the same buffer. The collected fractions were analysed byWestern blotting for rhamnogalacturonase as described above andL-arabinofuranosidase B (exo-B) activity by the method described by vander Veen et al (1991), since exo-B is known to co-elute withrhamnogalacturonase (Schols et al 1990a). To remove the exo-B therhamnogalacturonase containing fractions were dialysed against 5 mMpiperazine/HCl buffer pH 6.0 and reloaded on the same DEAE SepharoseFast Flow column and eluted with 1000 ml NaCl gradient (0-0.3 M) in thesame buffer. Further purification of rhamnogalacturonase involvedfractionation using a Pharmacia (Pharmacia, Uppsala Sweden) FPLC systemequiped with a MONO-P HR 20/5 column (Pharmacia) equilibrated in 20 mMpiperazine/HCL buffer pH 6.0. The column was eluted with 10%polybuffer74 (Pharmacia)/HCl pH 3.5. The fractions containing thehighest rhamnogalacturonase concentrations as determined by Westernblotwere pooled, the pH was brought to pH 6.0 with HCl and reloaded on theMONO-P HR 20/5 column. The column was eluted with 8% polybuffer74/HCl pH3.5. Further purification of rhamnogalacturonase was done by using aSuperose 12 column (dimensions 1.6*64 cm) equilibrated in 0.1 M sodiumacetate buffer pH 5.5 and 0.1 M NaCl. The column was eluted with thesame buffer. The collected fractions, containing the purifiedrhamnogalacturonase, were analysed by SDS-PAGE and Western blotting,resulting in one distinct band, showing cross reactivity with therhamnogalacturonase antibodies.

2.1.2 Determination of the N-terminal amino acid sequence of A.aculeatus rhamnogalacturonase

The pure rhamnogalacturonase fraction was used for determination of theN-terminal amino acid sequence of rhamnogalacturonase from A. aculeatusCBS 172.66, using the sequential degradation method of Edman and anApplied Biosystems Sequencer Model 475 with an on-line PTH-analyzermodel 120A. Although various amounts of purified protein and varyingconditions were employed, the N-terminal residue(s) of A. aculeatusrhamnogalacturonase could not be identified. It was concluded that A.aculeatus extracellular rhamnogalacturonase resists Edman degradation ofthe N-terminus, and consequently probably is modified at the N-terminus.Such modifications are rarely encountered in extracellular proteins,though some cases have been described, in which the modification isderived from rearrangement of an N-terminal glutamine residu, yielding apyroglutamic acid group. Thus, the N-terminal amino acid residu of A.aculeatus rhamnogalacturonase presumably is a glutamine.

2.1.3. Determination of amino acid sequences of internal regions of A.aculeatus rhamnogalacturonase

The purified rhamnogalacturonase fraction was used to generate fragmentsof the rhamnogalacturonase polypeptide by cleavage with CNBr essentiallyaccording to Gross and Witkop (as described in "Sequencing of Proteinsand Peptides", G. Allen, Laboratory Techniques in Biochemistry andMolecular Biology, Ed. T. S. Work and R. H. Burdon, 1981). The fragmentswere separated by HPLC, using a Bakerbond C4 wide pore column (5 μm;4.6*250 mm) (FIG. 2). The fractions corresponding to peaks 7 and 15 ofthe elution profile were named CNBr fragment #7 (sequence listing no.1), resp. CNBr fragment #5 (sequence listing no. 2), and were subjectedto amino acid sequence analysis according to the method of Edman (FIG.3, sequence listing no. 1 and no. 2). Another set of fragments of therhamnogalacturonase polypeptide was obtained by cleavage withEndoproteinase Lys-C (Boehringer, Mannheim), essentially as described byAitken et al. (1989). The fragments were separated by HPLC, using aBakerbond C4 wide pore column (5 μm; 4.6*250 mm) and selected fractionsEndolys#5 (sequence listing no. 4), Endolys#7 (sequence listing no. 3)were subjected to amino acid sequence analysis according to the methodof Edman (FIG. 3, sequence listing no. 3 and no. 4).

2.2. Isolation and characterization of cDNA of the A. aculeatus rhgAgene

All techniques used for the manipulation and analysis of nucleic acidmaterials were performed essentially as described in Maniatis et al.(1982), except where indicated otherwise.

2.2.1 Construction and screening of an expression library

A. aculeatus CBS 115.80 was grown in a 2.5 liter fermenter (workingvolume 2 liter) as described in chapter 1.1.4. on minimal medium (pH=6regulated with KOH) supplemented with 1% sucrose, 0.2% yeast extract and0.2% casamino acids. After 24 hours of growth cells were removed byfiltration on miracloth, washed twice in minimal medium and resuspendedin the original volume of minimal medium supplemented with 1% applepectin (brown ribbon apple pectin (degree of esterification 72.8%),obipectin, pH=4, regulated with KOH) as a carbon source. Stronginduction of the rhamnogalacturonase gene was observed in less then 6 hrafter the shift, as judged by Western analysis of the supernatant withthe rhamnogalacturonase-antiserum (FIG. 4). Therefore, total RNA wasisolated 3, 6 and 24 hr after shifting from sucrose medium to 1% applepectin using the guanidinium thiocyanate method and purified twice bycesium chloride density gradient centrifugation, essentially asdescribed by Maniatis et al. (1982). Total RNA isolates derived atdifferent moments in time were pooled and a polyA⁺ fraction (mRNA) wasisolated using a polyATtract mRNA isolation kit (Promega). This mRNAfraction was used for the construction of a cDNA library using a ZAPcDNA synthesis kit (Stratagene, La Jolla) according to the instructionsof the supplier, yielding cDNA fragments with a XhoI cohesive endflanking the poly-A region and an EcoRI adaptor at the other end. Theobtained cDNA fragments were used for directional cloning in the senseorientation in lambda ZAPII vectors (Stratagene, La Jolla), allowingexpression of β-galactosidase fusion proteins (Huse et al., 1988). Inorder to increase the sensitivity of the screening procedure, theinserts were excised from the lambda-vectors as phagemids by infectionwith helper phage R408 (Stratagene, La Jolla) and packaged phagemidparticles were isolated from the culture according to the instructionsof the supplier. The obtained mixture of pBluescript SK- phagemids wasused to infect E.coli BB4 (Stratagene, La Jolla) and cells were platedon LB-Amp selection plates. The obtained colonies were transferred tonitrocellulose filters (Schleicher & Sch uell, presoaked with a 10 mMIPTG solution); filters were placed on LB plates, containing ampicillinand 10 mM IPTG (colony side up), and were incubated for 3 hours at 37°C. to induce expression of β-galactosidase fusion proteins. The filterswere lifted from the plates and subjected to the following treatment:soaking for 3 minutes in 0.5 M NaOH and 8 M urea (cell lysis,denaturation of all proteins), followed by soaking for 3 minutes in 0.5M Tris/HCl,1.5 M NaCl (pH 7.5) (neutralization). The filters were washed3 times in standard TTBS and incubated withrhamnogalacturonase-antiserum as described by Burnette (1981). 2×10⁵colonies were screened using this procedure, among which 36 positivecolonies were identified. Double stranded phagemid DNA was purified from4 independent colonies that scored positive withrhamnogalacturonase-antiserum, and was characterized further at DNAlevel.

2.2.2 Sequence analysis of A. aculeatus rhgA cDNA clones

cDNA inserts were sequenced according to the method of Sanger using theSK primer (Stratagene, La Jolla), the T7 primer (Stratagene, La Jolla)and dedicated synthetic oligonucleotides. The insert of pUR7510 (FIG. 5)was completely sequenced in both directions, and was found to comprisethe entire coding region of the rhamnogalacturonase gene (rhgA, FIG. 6,sequence listing no. 5). Regions encoding amino acid sequencescorresponding to CNBr fragment #7, CNBr fragment #15, Endolys#5 andEndolys#7 (sequence listing no. 1, no. 2, no. 4 and no. 3 respectively)were encountered within the coding region, positively identifying thecloned cDNA as corresponding to A. aculeatus rhgA. An Escherichia coliDH5α strain containing this plasmid (CBS 238.92) was deposited at theCentraal Bureau voor Schimmelcultures (CBS) in Baarn, the Netherlands,on May 1th 1992.

2.3. Northern analysis of A. aculeatus RNA

A. aculeatus CBS 115.80 was cultured on 1% sucrose medium and shifted to1% apple pectin as a carbon source as described above. Total RNA wasisolated 0, 6 and 24 hr after shifting from sucrose medium to 1% applepectin using the guanidinium thiocyanate method, essentially asdescribed by Maniatis et al. (1982). 5 μg of each total RNA sample wasglyoxylated and subjected to Northern hybridization analysis using the1.2 kb KpnI fragment of pUR7510 as a probe. A single hybridizationsignal was obtained with samples isolated at 6 and 24 hr after shiftingto pectin, corresponding to a mRNA length of approximately 1500 bp. Nosignal was detected in RNA isolated from sucrose grown cells. Using rRNAas marker it was concluded that A. aculeatus rhgA mRNA has a length ofabout 1500 nucleotides. Since the cDNA inserts, constructed from mRNAafter induction of rhamnogalacturonase expression, were isolated bycross reactivity with rhamnogalacturonase antibodies after expression ofthe cNDA library and since the isolated cDNA inserts hybridise with mRNAafter induction of rhamnogalacturonase expression, the isolated cDNAinserts represent a part of the nucleotide sequence of the rhgA gene.

2.4 Number of rhgA and related genes in A. aculeatus and A. niger.

The 1.2 kb KpnI fragment of pUR7510, comprising part of the codingregion of the A. aculeatus rhgA gene, was labeled according to astandard random primer labeling protocol and used as a probe forSouthern hybridization analysis of restriction enzyme digests of A.aculeatus and A. niger N400 (CBS 120.49) genomic DNA, to establish thenumber of rhgA (or related) genes that are present in the genome of A.aculeatus and A. niger N400. Using standard methods and stringentconditions for hybridization and washing of the blot, the hybrizationpatterns of the various restriction enzyme digests of A. aculeatusgenomic DNA revealed that only a single copy of the rhgA gene is presentin the A. aculeatus genome. However, using less stringent conditions atleast one related gene was detected in A. aculeatus and at least threerelated genes in A. niger N400.

2.5. Isolation of the rhgA gene from A. aculeatus genomic DNA

Since the cDNA inserts described above were derived from isolated mRNA,they do not contain possibly occurring introns. In order to isolate theentire DNA sequence encoding rhamnogalacturonase and its expressionsignals present on the genome, isolated cDNA inserts comprising part ofthe coding region of the rhgA gene were used as a probe to screen agenomic library of A. aculeatus.

2.5.1 Construction of a genomic library of A. aculeatus DNA

High molecular weight genomic DNA of A. aculeatus CBS 115.80 wasisolated as described by De Graaff et al. (1988). It was partiallydigested with MboI and the fragments were size fractionated on a sucrosegradient before cloning into the BamHI site of lambda-EMBL4 (Karn J. M.et al 1980). The library comprises 2.9×10⁶ independent clones with aninsert size range of 8 to 21 kb and was constructed by ClontechLaboratories Inc.

2.5.2 Isolation of lambda clones comprising the A. aculeatus rhgA gene

The obtained library of A. aculeatus genomic DNA in lambda-EMBL4 wasscreened using the 0.6 kb XhoI fragment of pUR 7510, comprising part ofthe coding region of the A. aculeatus rhgA gene, as a probe. Thefragment was labelled according to a standard random primer labelingprotocol. Approximately 20×10³ plaques were tested in duplo (duplicatefilters from each plate) according to standard methods (Maniatis et al.,1982) using E.coli LE392 as plating bacteria. The total length of theinserts contained within the analyzed plaques is equivalent to about 16times the size of the A. aculeatus genome. Hybridization was performedin 6*SSC, 0.5% SDS, 5*Denhardt solution, 20 μg single stranded herringsperm DNA at 65° C. (Maniatis et al, 1982). Filters were washed threetimes for 15 minutes in 2*SSC, 0.1% SDS at 65° C. Eighteen plaques,which were also found to hybridize with the duplicate set of filters,were subjected to a rescreening procedure. Using standard procedures DNAwas isolated from four independent plaques that scored positive in therescreening procedure.

2.5.3. Physical mapping of genomic k-clones comprising part of the A.aculeatus rhgA gene

The inserts of these four positive clones were mapped by Southernhybridization analysis of single and combined digestions with therestriction enzymes BamHI, BglII, EcoRI, HindIII, SalI, SmaI and XhoIusing the 1.2 kb KpnI fragment of pUR7510, comprising part of the codingregion of the A. aculeatus rhgA gene, as a probe. The fragment waslabelled according to a standard random primer labeling protocol.Combination of the resulting data with known locations of the KpnI andSmaI sites within the cDNA sequence (derived from pUR7510) led to theidentification of a 3.9 kb BamHI-SalI fragment which comprises theentire A. aculeatus rhgA gene (FIG. 7).

2.5.4. Sequencing the A. aculeatus rhgA gene

The 3.9 kb BamHI-SalI fragment comprising the entire A. aculeatus rhgAgene was subcloned in pBluescript SK+(Stratagene, La Jolla), yieldingpUR 7511 (FIG. 8). An Escherichia coli DH5α strain (containing thisplasmid (CBS 239.92) was deposited at the Centraal Bureau voorSchimmelcultures (CBS) in Baarn, the Netherlands, on May 1th, 1992. Byfurther mapping of restriction enzyme sites in the insert of pUR7511, a2.5 kb HindIII fragment comprising the entire coding region of the rhgAgene was identified. The sequence of this fragment was determined bysequencing the entire fragment in both directions according to themethod of Sanger, using T3-primer (Stratagene, La Jolla), T7-primer(Stratagene, La Jolla) and dedicated synthetic primers (FIG. 9, sequencelisting 7). Additional sequence information for the region upstream ofthe rhgA gene was also obtained from pUR7511. Comparison of the genomicDNA sequence of the rhgA gene with the cDNA sequence unambiguouslyidentified the position and the size of three introns (FIG. 9, sequencelisting 7).

2.6 Isolation of the rhgI and rhgII genes from A. niger genomic DNA

2.6.1 Isolation of lambda clones comprising the A. niger rhgI and rhgIIgene

The library of A. niger N400 genomic DNA in lambda-EMBL4 (Harmsen et al1990) was screened using the 2.5 kb HindIII fragment of pUR7511,comprising the coding region of the A. aculeatus rhgA, as a probe. Thefragment was labeled according to a standard random primer labelingprotocol. Approximately 40×10³ plaques were tested in duplo (duplicatefilters from each plate) according to standard methods (Maniatis et al.,1982) using E.coli LE392 as plating bacteria. The total length of theinserts contained within the analyzed plaques is equivalent to about 25times the size of the A. niger genome. Hybridization was performed in6*SSC, 0.5% SDS, 5*Denhardt solution, 20 μg/ml single stranded herringsperm DNA at 58° C. (Maniatis et al, 1982). Filters were washed threetimes for 15 minutes in 2*SSC, 0.1% SDS at 58° C. Eight plaques whichwere found to hybridize also in the duplicate set of filters, weresubjected to a rescreening procedure. Using standard procedures, DNA wasisolated from five independent plaques that scored positive in therescreening procedure.

2.6.2. Physical mapping of genomic k-clones comprising part of the A.niger rhg gene

The inserts of these five positive clones were mapped by Southernhybridization analysis of single and combined digestions with therestriction enzymes BamHI, BglII, EcoRI, HindIII, SalI, PstI and HindIIusing the 2.5 kb HindIII fragment of pUR7511, comprising the codingregion of the A. aculeatus rhgA gene, as a probe. The fragment waslabelled according to a standard random primer labeling protocol.Analysis of the resulting data revealed two classes of phages (class Iand class II) which are distinguished by hybridization intensity andrestriction pattern. Four of the five phages that were analysed belongto the stronger hybridyzing class (I), one phage to the less hybridizingclass (II). The corresponding genes (respectively rhgI and rhgII) werecharacterized by restriction enzyme analysis (FIG. 10). Homologies withthe A. aculeatus rhgA protein are 80% and 70% for the rhgI and rhgIIproteins respectively, as deduced from a 1500 bp part of the codingregion, which was sequenced.

2.6.3. Number of rhgI and closely related genes in A. aculeatus and A.niger

A 2.5 kb BamHI- HindIII fragment, comprising the coding region of the A.niger rhgI gene, was labeled according to a standard random primerlabeling protocol and used as a probe for Southern hybridizationanalysis of restriction enzyme digests of A. aculeatus and A. niger N400genomic DNA, to establish the number of rhgI (or closely related) genesthat are present in the genome of A. aculeatus and A. niger N400. Usingstandard methods and non-stringent conditions for hybridization andwashing of the blot, the hybrization patterns of the various restrictionenzyme digests of A. aculeatus and A. niger genomic DNA revealed that atleast two related genes were detected in A. aculeatus and A. niger (FIG.11).

EXAMPLE 3

Overproduction of Aspergillus aculeatus rhgA gene in a suitableAspergillus aculeatus acceptor strain under the control of regulatoryelements of the Aspergillus aculeatus rhgA gene

In order to construct an A. aculeatus strain which is capable ofoverproducing rhamnogalacturonase, multiple copies of the A. aculeatusrhgA gene were introduced into a suitable acceptor strain byco-transformation with the A. niger pyrA gene.

3.1 Construction of a suitable A. aculeatus host strain

A. aculeatus NW215 (cspA1, fwnA1, pyrA4) was used as an acceptor strainin transformation experiments. The cspA1 mutation results in shortconidiophores and fwnA1 mutation results in yellowish brown spores. ThepyrA mutation causes uridine requirement for growth, and is utilized forthe introduction of multiple copies of the rhgA gene in cotransformationexperiments. For the construction of A. aculeatus NW215, mutations wereinduced in condiospores of A. aculeatus CBS 115.80 by UV illuminationusing the method described by Bos (1987). Conidiospore survival variedfrom 20 to 57% in the different experiments. A subset of mutagenizedspores was selected for uridine deficiency using 5-fluoro orotic acidand standard procedures to screen for a pyrA mutation, yielding amongstothers A. aculeatus strain NW 212 (pyrA4).

Another subset of mutagenized spores was selected for shortconidiophores. A. aculeatus strain NW210 (cspA1), one of the resultingstrains, was again mutagenized to obtain colour mutations. A. aculeatusstrain NW213 (cspA1, fwnA1), one of the resulting strains producingyellowish brown spores, was again mutagenized and selected forauxotrophic mutations using a standard filtration enrichment procedure(Uitzetter et al., 1986). Several of the resulting mutant strainscarried auxotrophic mutations, among which A. aculeatus strain NW214(cspA1, fwnA1, lysB2) and A. aculeatus strain NM216 (cspA1, fwnA1,lysA1) were identified. Two crosses were performed: 1) A. aculeatusstrain NW212 with A. aculeatus strain NM214 and 2) A. aculeatus strain212 with A. aculeatus strain NW216, using the method described by Bos(1987). From the isolated heterozygous diploid colonies a few sporeswere streaked directly onto CM-benomyl plates for haploidization. Amongthe resulting recombinant strains A. aculeatus strain NW215 (cspA1,fwnA1, pyrA4) and A. aculeatus strain NM217 (cspA1, fwnA1, pyrA4, lysA1)were found.

3.2 Co-transformation of A. aculeatus using pyrA as selectable marker

A. aculeatus strain NW215 was co-transformed with mixtures of twodifferent DNA fragments in various ratios using standard techniques (e.gGoosen et al., 1987). The two fragments used were the 3.8 kb XbaIfragment of A. niger N400, comprising the entire A. niger pyrA geneincluding functional promoter (Goosen et al., 1987), and a 3.9 kbBamHI-SalI fragment of A. aculeatus CBS 115.80, comprising the entirerhgA gene and flanking sequences (FIG. 8). The two plasmids were used ina ratio of 1:20 for pyrA:rhgA.

3.3. Identification and analysis of multicopy transformants

Transformed strains were selected by their ability to grow in theabsence of uridine. A selected number of transformants was analysed forcotransformation. They were subsequently grown in a medium containingper liter 1.9 g NH₄ Cl, 0.26 g KCl, 0.76 g KH₂ PO₄. 0.26 g MgSO₄.7 H₂ O) adjusted to pH 6.0 with KOH, 1% sugar beet pulp and 1 ml of a1000×concentrated Visniac trace elements solution (Visniac and Santer1957). Incubation was carried out for 48 hours in 250 ml shake flasksshaking at 30° C. after which the culture was filtrated throughmiracloth. The culture filtrate was analysed on a SDS-PAGE gelsystemfollowed by Western blotting as described above. Transformants which hadincorporated one or more additional copies of the A. aculeatus rhgA genewere identified by quantitative Western analysis of the culturesupernatant using the rhamnogalacturonase-antiserum (FIG. 12). Thus,overproduction of A. aculeatus rhgA was achieved by the introduction ofadditional copies of the A. aculeatus rhgA gene in the A. aculeatusgenome in at least 10 strains of A. aculeatus which paves the way forthe production of rhamnogalacturonase at an economically feasible scale.Determination of the copynumber of rhgA genes in two transformants (67and 75) by Southern analysis revealed that about 5-8 additional copiesof the rhgA gene have been introduced in both transformants of A.aculeatus . Transformant 75 and the wild type strain were grown in 250ml shake flasks on minimal medium and 1% sucrose as described above.After 24 hours of incubation 1% apple pectin was added. After another 24hours the culture was filtered over miracloth and the filtrate wasconcentrated and analysed for rhamnogalacturonase activity using the DNSmethod to demonstrate reducing sugars. A three fold increase (OD 0.64and 1.94 for wild type strain and transformant respectively) inrhamnogalacturonase activity was observed.

These filtrates were also used in a apple mash experiment as describedin chapter 1.2.2. Viscosity reduction was measured after 60 minutes ofincubation and was 89%, 90.5% and 93% after incubation with BiopectinaseLQ, Biopectinase LQ+wild type filtrate and Biopectinase+transformantfiltrate respectively. These results clearly show that overproduction offunctional rhamnogalacturonase, capable of improving viscosity reductionin hot mash application trials, can be achieved by introduction ofadditional copies of the A. aculeatus rhg gene in the A. aculeatusgenome.

EXAMPLE 4

Overexpression of the Aspergillus aculeatus rhgA gene in Aspergillicontrolled by other regulatory elements than those derived from theAspergillus aculeatus rhgA gene

To further increase the production level of rhamnogalacturonase,alternative expression systems can be employed. For this purpose thecoding part of the rhgA gene can be detached from the upstream controlregions (comprising a.o. the promoter), and placed under the control ofother promoters derived from Aspergillus strains by fusion at theATG-translational start signal. Promoters directing the expression ofthe following genes can be used: the xylanase (xylA) gene of A. nigervar. awamori CBS 115.52 (xylA promoter), the glucoamylase (glaA) gene ofA. niger CBS 120.49 (glaA promoter) and theglyceraldehyde-3-phosphatedehydrogenase (gpdA) gene of A. nidulans (gpdApromoter).

4.1 xylA promoter

The 3.0 kb KpnI fragment of plasmid pAW14B (described in patentapplication WO 91/19782, Van Gorcom et al., 1991) was inserted in theKpnI site of pTZ19R, yielding pUR2930 (FIG. 13). This plasmid was usedas a template in a first PCR reaction (PCR1) using primer XYLNSI(5'-CGAGGTTGTTCAAGCGT-3', sequence listing no. 9) capable of hybridizingupstream of the NsiI site in the xylA promoter region of pUR2930, and ahybrid primer XYLRHG (5'-AGAAGGAAAAGAGCACGCATGATGATTGAAGAAAGCT-3',sequence listing no. 10) capable of hybridizing just upstream of theATG-translational start signal of the xylA gene and fusing it to theregion of the rhgA gene just downstream of the translational startsignal at the ATG codon. In a second PCR reaction (PCR2) using pUR7511as a template and a primer RHGXYL (5'AGCTTTCTTCAATCATCATGCGTGCTCTTTTCCTTCT-3', sequence listing no. 11),complementary to XYLRHG, and a primer RHGKPN (5'-ATCATGTTCCCACTGGC-3',sequence listing no. 12), capable of hybridizing just downstream of theKpnI site of the A. aculeatus rhgA gene, the sequence of the xylA geneimmediately upstream of the ATG translational startsignal was fused tothe first section of the rhgA gene. The fragments resulting from PCR1and PCR2 were mixed and subjected to another round of PCR amplification(PCR3). The fragment resulting from PCR3 is digested with NsiI and KpnIand used in a ligation reaction with the 2.4 kb BamHI-NsiI fragment ofpUR2930 and the 5.4 kb KpnI-BamHI fragment of pUR7511 (partiallydigested with KpnI). The resulting plasmid pUR 7512 comprises the A.aculeatus rhgA gene fused to the A. niger var. awamori xylA promoter atthe ATG-translational startsignal (FIG. 14).

This plasmid and suitable fragments thereof can be used together withthe 3.8 kb XbaI-fragment of the A. niger pyrA gene in co-transformationsof A. aculeatus strain NW215, essentially as described section 3.2Transformants with a PYR⁺ phenotype can be screened for the presence ofmultiple copies of the rhgA gene by Southern hybridisation analysis,which is facilitated by the differences in the length of fragmentsgenerated by restriction enzyme digestions between the native copy ofthe rhgA gene and the copies behind the xylA promoter that are newlyintroduced, or by Western analysis of the culture supernatant using therhamnogalacturonase-antiserum. Alternatively, the amdS gene of A.nidulans can be inserted in pUR7512, and used in transformationexperiments for selection of transformants containing multiple copies ofthe rhgA gene. Transformants containing multiple copies of the newlyintroduced A. aculeatus rhgA gene behind the A. niger var. awamoripromoter can be grown in media that induce increased transcriptionlevels from the xylA promoter, for example media containing wheat branor xylan as described in patent application WO 91/19782 (Van Gorcom etal., 1991). Similarly, A. niger strains containing genetic markers, forexample pyrA⁻ routants of A. niger . N400 can be used incotransformations in order to introduce multiple copies of the A.aculeatus rhgA gene under the control of the xylA promoter in thesestrains. Moreover, following the same approach, plasmid pUR7512 andsuitable fragments or plasmids derived therefrom, can be used for theintroduction of multiple copies of the A. aculeatus rhgA gene under thecontrol of the xylA promoter into strains of A. aculeatus or otherspecies of the genus Aspergillus, for example A. oryzae, A. japonicus,A. sojae, A. tubigensis, A. awamori, A. nidulans, etc.

As a further example a 5.4 kb BamHI-SalI fragment of plasmid pUR7512,comprising the rhgA gene of A. aculeatus under the control of the xylApromoter from A. niger var. awamori (FIG. 14) was introduced in A. nigervar. awamori CBS 115.52. To this end a pyrA- variant of A. niger var.awamori CBS 115.52 was constructed using UV- mutagenesis and screeningon fluoroorotic acid plates (3, 10 E6 spores were irradiated for 90seconds at 20 erg/mm2/sec with UV radiation, yielding 44% survival).Within the obtained group of pyr- variants two complementation groupscould be identified by transformation with the 3.8 kb XbaI fragment ofA. niger N400, comprising the entire A. niger pyrA gene and functionalpromoter (Goosen et al. 1987). A pyr A- variant of A. niger var. awamoriCBS 115.52 that could be complemented by the A. niger pyr A gene wasidentified and named strain NW208. Multiple copies of the fusionconstruct comprising the A. aculeatus rhgA gene under the control of thexylA promoter were introduced in this strain by co-transformation of a5.4 kb BamHI-SalI fragment of plasmid pUR7512 (FIG. 14) comprising therhgA gene of A. aculeatus under the control of the xylA promoter from A.niger var. awamori and the 3.8 kb XbaI-fragment of the A. niger pyr Agene, essentially as described in chapter 3.2. Six transformants with apyr A+phenotype were screened on rhamnogalacturonase activity by Westernanalysis according to the following method: Strains were grown inbaffled shake flasks (500 ml) with 200 ml synthetic media (pH 6.5 withKOH) after inoculation with 10 E6 spores/mi. The medium had thefollowing composition (AW medium):

    ______________________________________                                        sucrose               10     g/l                                              KCl                   0.52   g/l                                              MgSO.sub.4.7H.sub.2 O 0.49   g/l                                              ZnSO.sub.4.7H.sub.2 O 22     mg/l                                             MnCl.sub.2.4H.sub.2 O 5      mg/l                                             CaCl.sub.2.6H.sub.2 O 1.7    mg/l                                             NaH.sub.2 MoO.sub.4.2H.sub.2 O                                                                      1.5    mg/l                                             NaNO.sub.3            6.0    g/l                                              KH.sub.2 PO.sub.4     1.52   g/l                                              Yeast extract         1.0    g/l                                              H.sub.3 BO.sub.3      11     mg/l                                             FeSO.sub.4.7H.sub.2 O 5      mg/l                                             CuSO.sub.4.5H.sub.2 O 1.6    mg/l                                             Na.sub.2 EDTA         50     mg/l                                             ______________________________________                                    

Incubation took place at 30° C., 125 rpm for 24 hours in a Mk Xincubator shaker. After growth cells were collected by filtration (0.45μm) washed twice with AW medium without sucrose and yeast extract (saltsolution), transferred to 500 ml shake flasks and resuspended in 100 mlsalt solution to which xylose was added to a final concentration of 10g/l (induction medium). After 24 hours biomass was removed by filtrationover miracloth and rhamnogalacturonase was detected by Western analysisas described above (FIG. 15). From FIG. 15 it is a clear thatrhamnogalacturonase can be efficiently produced in A. niger var. awamoriusing the xylA promoter. Strong overexpression was found intransformants 3 and 5. Activity of A. aculeatus rhamnogalacturonase,produced by A. niger var. awamori under the control of the xylApromoter, on Modified Hairy Regions was done as described in chapter1.2.1 (10 μl of filtrate of transformant 3 (see above) added to MHR) andanalysed with the Dionex system (FIG. 16). From FIG. 16 it is clear thatrhamnogalacturonase produced by A. niger var. awamori under the controlof the xylA promoter is active on isolated Modified Hairy Regions.Application trials as described in chapter 1.1.2. were performed withBiopectionase LQ (500 g/ton) in the presence and absence of 3 ml offiltrate of A. niger var awamori transformant 3, cultured as describedabove. Juice yield was measured after 2 hours of incubation and a 10%increase in yield at equal brix was observed in the presence ofrhamnogalacturonase.

Thus high expression levels of functional A. aculeatusrhamnogalacturonase were achieved in A. niger var. awamori under thecontrol of the A. niger var. awamori xylanase promoter.

4.2 glaA promoter

An approach similar to that outlined in section 4.1 can also be followedfor the construction of plasmids in which the A. aculeatus rhgA gene hasbeen fused to the promoter of the A. niger glaA gene at theATG-translational startsignal, yielding plasmids essentially asdescribed in patent application WO 91/19782 (Van aGorcom et al., 1991).In this case pAN52-6 (Van den Hondel et al, 1991) can be used as atemplate in PCR reactions providing the functional glaA promoter.

The resulting plasmids or suitable fragments thereof can be used togenerate transformants containing multiple copies of the A. aculeatusrhgA gene under the control of the A. niger glaA promoter in strains ofspecies of the genus Aspergillus carrying genetic markers, essentiallyas outlined in section 4.1 of this example.

4.3 gpdA promoter

An approach similar to that outlined in section 4.1 can also be followedfor the construction of plasmids in which the A. aculeatus rhgA gene isfused to the promoter of the A. nidulans gpdA gene at theATG-translational startsignal, yielding plasmids essentially asdescribed in patent application WO 91/19782 (Van Gorcom et al., 1991).For this purpose pAN52-1 (Punt et al., 1987) can be used as a templatein PCR reactions to generate a fragment comprising the gpdA promotersequences up to the ATG-translational startsignal fused to the rhgAcoding region. The resulting plasmids or suitable fragments thereof canbe used to generate transformants containing multiple copies of the A.aculeatus rhgA gene under the control of the A. nidulans gpdA promoterin strains of species of the genus Aspergillus carrying genetic markers,essentially as outlined in section 1. of this example.

EXAMPLE 5

Production of Aspergillus aculeatus rhamnogalacturonase in yeast

5.1 Introduction

For the production of A. aculeatus rhamnogalacturonase in yeasts,vectors can be constructed in which the sequences encoding the matureAspergillus aculeatus rhamnogalacturonase protein are fused to yeastregulatory sequences for the transcription of the gene. If secretion ofthe rhamnogalacturonase protein is desired, functional yeast signalsequences can be added to the coding sequence of the rhgA gene. Sinceyeasts may not be capable of correct removal of the introns from theprimary RNA transcript of the A. aculeatus rhgA gene, the introns shouldnot be present in these constructions. The cDNA fragment of the rhgAgene, present in pUR7510, can serve as a base for such constructions.

Efficient production and secretion by yeast of an Aspergillus gene waspreviously described for the xylanase gene of Aspergillus niger var.awamori in patent application WO 91/19782 (Van Gorcom et al., 1991).Production and secretion of the xylanase was achieved by fusion of thepromoter from the Saccharomyces cerevisiae GAL7 gene, and the signalsequence of the Saccharomyces cerevisiae SUC2 gene encoding theinvertase protein, to the mature xylanase gene from which the intron wascorrectly removed. Production of the Aspergillus aculeatusrhamnogalacturonase protein in yeast can be accomplished by a functionalfusion of the GAL7-promoter and invertase signal sequence from yeast tothe mature, intronless, rhgA gene. Such constructions can beincorporated in either autonomously replicating yeast vectors, oralternatively, in yeast vectors that are capable of integration in theyeast genome in single or multiple copies. The levels of expression ofthe A. aculeatus rhgA gene directed by such constrictions can be furtherimproved by adjustment of the codon usage of the A. aculeatus rhgA geneaccording to the codon preferences known for yeasts.

5.2 Expression in Saccharomyces cerevisiae of the rhamnogalacturonaseprotein using autonomously replicating vectors

The expression plasmid pUR2904 (FIG. 17), used for the secretion of theAspergillus niger var. awamori xylanase protein by Saccharomycescerevisiae (Van Gorcom et al., 1991), can serve as basis for theexpression of the A. aculeatus rhamnogalacturonase protein in yeast.pUR2904 is an E. coli-S. cerevisiae shuttle plasmid which contains acorrect fusion of the coding sequences of the S. cerevisiae invertasesignal sequence and the mature A. awamori xylanase, under control of thepromoter sequences of the S. cerevisiae GAL7-gene. Furthermore, thisplasmid contains the replication origin of the yeast 2-micron plasmid,the S. cerevisiae LEU2-gene, and the SalI-EcoRI fragment of pBR322containing the ampicillin resistance gene and the MB1 replicationorigin.

The sequences coding for the mature rhamnogalacturonase protein, aspresent in pUR7510, can be fused to the GAL7-promoter and invertasesignal sequence, as present in pUR2904, by using the same approach asdescribed for the fusion of the rhgA gene to the xylA promoter insection 4.1. Here, pUR2904 can be used as a template in a first PCRexperiment using primer GALBGL (5'-GAAGTTAGATCTAGCTATACT-3', sequencelisting no. 13) capable of hybridizing at the beginning of theGAL7-promoter on the BglII-site, and primer INVRHG(5'-CAACACTGCCAGAGAGTTGCGCAGATATTTTGGCTGCAA-3', sequence listing no. 14)serving the correct fusion of the invertase signal sequence to themature rhamnogalacturonase protein. In a second PCR experiment pUR7510can be used as a template using primer RHGINV(5'-TTGCAGCCAAAATATCTGCGCAACTCTCTGGCAGTGTTG-3', sequence listing no.15), complementary to INVRHG, and primer RHGEND(5'-CCCAAGCTTCAATCAACTACTAGCCTGCCAAGGCA-3', sequence listing no. 16)capable of hybridizing to the end of the coding sequence of the rhgAgene, and adding additional translational stopcodons and a HindIII-siteto the 3' end of it. In the third PCR-experiment the two products arefused and after digestion with BglII and HindIII the 1662 bp fragmentcan be ligated to DglII--HindIII digested pUR2904 resulting in plasmidpUR7513 (FIG. 18). Plasmid pUR7513 will then contain the signal sequenceof the Saccharomyces cerevisiae invertase, correctly fused to the codingsequences of the mature Aspergillus aculeatus rhamnogalacturonaseprotein, under transcriptional control of the Saccharomyces cerevisiaeGAL7 promoter sequences. Expression of the rhamnogalacturonase proteinby yeast can then be achieved by transformation of a suitable S.cerevisiae strain with pUR2913, and growing leu+transformants in mediacontaining galactose as a carbon source.

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We claim:
 1. A method of detecting a ripening form of a polypeptidewhich cross reacts with an antibody which specifically binds to anantigenic determinant of rhamnogalacturonase comprising the stepsof:adding said antibody to a sample containing said polypeptide;incubating said antibody with said sample such that said antibody bindsto said polypeptide; and detecting the presence of said polypeptidebound to said antibody.
 2. A method of detecting an organism whichproduces or secretes a polypeptide which cross reacts with an antibodywhich specifically binds to an antigenic determinant ofrhamnogalacturonase comprising the steps of:incubating the antibody withan organism suspected of producing or secreting said polypeptide underdetecting said organism by determining the presence of said conditionswhereby said polypeptide binds to said antibody; and polypeptide boundto said antibody.
 3. A method of isolating a ripening form of apolypeptide which cross reacts with an antibody which specifically bindsto an antigenic determinant of rhamnogalacturonase comprising the stepsof:adding the antibody to a sample containing said polypeptide;incubating said antibody with said sample such that said antibody bindsto said polypeptide; and isolating said polypeptide.
 4. A method ofisolating an organism which produces or secretes a polypeptide whichcross reacts with an antibody which specifically binds to an antigenicdeterminant of rhamnogalacturonase comprising the steps of:incubatingthe antibody with an organism suspected of producing or secreting saidpolypeptide under conditions whereby said polypeptide binds to saidantibody; detecting the presence of said polypeptide bound to saidantibody; and isolating said organism.